Particulate drug delivery

ABSTRACT

Methods for efficient preparation of drug-polymer (or oligomer) conjugates which are useful in the preparation of particles, including microparticles and nanoparticles, for delivery of the drug in vivo for therapeutic applications. The invention additionally provides certain drug-polymer and drug-oligomer conjugates which are useful in the preparation of particles for delivery of the drug in vivo. The invention also provides nanoparticles of this invention prepared by nanoprecipitation using drug-polymer/oligomer conjugates of the invention. 
     The drug conjugates are formed during polymerization of the polymer or oligomer in which the drug is employed as an initiator of the polymerization of the monomers which form the polymer and/or oligomer. More specifically, the drug conjugates are formed by ring-opening polymerization of cyclic monomers in the presence of an appropriate ring-opening polymerization catalyst and the initiator (the drug). The method is particularly useful for formation of polymer/oligomer conjugates with drugs and other chemical species containing one or more hydroxyl groups or thiol groups.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application60/892,834, filed Mar. 2, 2007, which is incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

Polymer nanoparticles (NPs) play an important role in drug delivery andare particularly useful for delivery of chemotherapy drugs. For clinicalapplications, the control of nanoparticle size and surface morphologyare important. Other aspects of the design of particulate systems canalso be important for the use of nanoparticles as delivery systems invivo. It is preferred that drug loading in the polymeric nanoparticle isreasonable high for improved efficacy. This is particularly importantfor enhanced effectiveness of nanoparticles in cancer therapy¹⁻³

High drug loading^(4,5) decreases manufacturing cost and increasespatient compliance by reducing the dose needed for each administration.In addition, drug molecules in nanoparticle delivery vehicles preferablyremain substantially encapsulated in the polymeric nanoparticles onadministration to a patient to be released in a sustained manner overtime or after they accumulate at a desired location. More specificallyfor nanoparticle applications to cancer therapy, it is important thatanticancer agents remain encapsulated with little or no drug releasewhile in the vasculature and release the anticancer agent only after thenanoparticles extravasate to tumor tissues.

Well-controlled drug release has been realized only in a limited numberof drug delivery systems, most of which are liposomes.⁶ Currently, thereare about 10 liposomal delivery vehicles approved for clinicalapplications of which only one, Abraxane, an albumin bound paclitaxelnanoparticle with size ˜130 nm, is a polymeric nanoparticulate deliveryvehicle.^(6,7) Abraxane,^(8, 9) appears to contain a large quantity oflipids on the surface of albumin nanoparticles which gives themliposome-like properties in regulating drug release. To date nopolyester based nanoencapsulates are approved for clinical cancertreatment.

In a recent study using poly(ethyleneglycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) to nanoencapsulatedocetaxel for in vivo prostate cancer treatment (Farokhzad, O. C.;Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie,J. P.; Langer, R. Proc. Nat'l Acad. Sci. (USA) 2006, 103, 6315-6320),difficulties were experienced in controlling formulation parameters suchas drug loading and encapsulation efficiency. The encapsulationefficiency, which depends on various parameters, including solvents,type of polymers, polymer molecular weights, and the drugs to beencapsulated, varied from batch to batch and was usually less than 80%.Drug loading was also typically lower than 10%. In many cases, only 1%of drug loading could be achieved. NPs with more than 5% of drug loadingsometimes contained undesired large aggregates (>1 micron), which waspresumably due to the aggregation of the non-encapsulated drugmolecules. Particles with mixed sizes and wide distributions can lead tocomplex biodistribution and pharmacokinetic responses in vivo.

In liposome delivery systems, drug molecules are encapsulated in thecore of the liposome and are, thus, separated from the externalenvironment by lipid bi-layers which prevent leakage of encapsulateddrug molecules. In contrast, polymer nanoencapsulates (polymericnanoparticles) have no such regulating mechanism to prevent the unwantedleaking of therapeutic molecules during circulation. Significant burstrelease effects are one of the greatest challenges to overcome in theapplication of polymeric nanoparticles in vivo for drug delivery. In avehicle with significant burst release effect, poorly encapsulated drugmolecules on or near the surface of nanoparticles can quickly diffuseinto solution and may lead to significant toxicity in vivo.¹⁰ Burstrelease is especially severe when drug loading exceeds the encapsulationthreshold of the polymer where there can be a significant amount of drugmolecules precipitated on the surface of the nanoparticle.

Nanoencapsulates (NE) usually display a biphasic drug releasepattern¹⁰⁻¹² with as high as 40-80% of the encapsulated drug moleculesburst released during the first several or tens of hours.¹⁰ After thefirst 24 to 48 hours, drug release becomes significantly slower due tothe increased diffusion barrier for drug molecules buried more deeply inpolymer nanoparticles. When these semi- or even completely emptynanoparticles eventually arrive and accumulate at the site where theyare needed (e.g., tumor tissue), they usually have little or noremaining therapeutic efficacy.^(6, 13)

It is extremely difficult to achieve high drug loading with highencapsulation efficiency in polymeric nanoencapsulates. Theencapsulation efficiency not only depends on the type, molecular weightand properties of the polymers used, but is also significantly affectedby the chemical and physical properties of therapeutic molecules. Forexample, lower molecular weight polymers tend to exhibit lowerencapsulation efficiency than higher molecular weight polymers.Hydrophilic molecules (e.g., doxorubicin) cannot be readily encapsulatedinto polymeric nanoparticles (Grovender T. et al. (1999) J. ControlledRelease 57(2) 171-185). In all nanoencapsulates so far developed, drugloading (the weight percentage of drug in polymer nanoparticles) andencapsulation efficiency (percentage of drug encapsulated relative tototal amount of drugs applied) vary dramatically from system to systemand from batch to batch. For hydrophobic small molecules such aspaclitaxel (Ptxl) or docetaxel (Dtxl), it is common that nanoparticleloading is in a range of 1 to 5 wt % and encapsulation efficiency variesfrom ˜20- to 80%.^(10, 14)

Another problem encountered in nanoparticle drug encapsulation isundesirable particle heterogeneity. Nanoprecipitation of polymer anddrugs, such as chemotherapeutics, frequently gives multimodaldistributions as measured by dynamic light scattering, ranging from ˜100nm to 1 μm or higher. Particle heterogeneity may result becauseencapsulation involves distinct chemical species, a polymer and a drugmolecule, with distinct molecular weights, flexibility and rigidity,hydrophobicity and tendencies toward forming crystals. Therefore it islikely the polymer and the drug molecule would tend to self-aggregateduring nanoprecipitation leading to particle heterogeneity.

Nanoparticle materials exhibiting multimodal distributions are usuallytreated as having a different degree of aggregation of smallnanoparticles with identical composition. However, this assumption maynot always be correct. In a recent investigation on the effect ofdocetaxel loading at 1%, 5% and 10% on resulting PEG-b-PLGA nanoparticlesize distributions (Cheng, J. et al. Formulation of functionalizedPLGA-PEG nanoparticles for in vivo targeted drug delivery. Biomaterials28, 869-76 (2007)), polydispersity of the particle preparationsincreased with docetaxel concentration from 0.154 for 1% loading to0.203 for 5% loading and 0.212 for 10% loading. The size distribution ofthe nanoparticles exhibited a biphasic trend with a smaller diameterparticle distribution accompanied by a distribution of larger diameterparticles. The distribution corresponding to the smaller particles didnot shift with the increase of drug concentration. The larger diameterlocus of the two size distributions shifted higher as the drug loadingincreased (the size increasing from ˜300 nm to ˜1200 nm). Since the onlydifference between these formulations is the amount of drug loading, asignificant amount of the nanoparticles formed may be due to aggregationof unencapsulated docetaxel due to its poor water solubility. In thiswork and that of others (Avgoustakis, K. et al. PLGA-mPEG nanoparticlesof cisplatin: in vitro nanoparticle degradation, in vitro drug releaseand in vivo drug residence in blood properties. J. Controlled Release79, 123-135 (2002)) on nanoprecipitation using polylactide anddocetaxel, biphasic particle distributions were almost always observed.

It is desirable to develop a methodology to circumvent thesedifficulties, which will provide NPs with batch-to-batch consistency inencapsulation efficiency and drug loading. This invention provides asimple, one-step strategy for the preparation of drug-polymer (anddrug-oligomer) conjugates which can be formed into nanoparticles with100% encapsulation efficiency and predetermined drug loading. Thenanoparticles formed by the methods herein employing drug conjugates arecall nanoconjugates herein to distinguish over nanoencapsulates (NE).Further, the method of this invention can be broadly applied to providepolymer and oligomer conjugates of a variety of useful chemical species(bioactive species, drugs, reagents, diagnostics, contrast agents,reporter molecules, dyes, etc.) for the preparation of particulatedelivery systems.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a one-step method forefficient preparation of certain drug-polymer (or oligomer) conjugateswhich are useful in the preparation of particles, includingmicroparticles and nanoparticles, for delivery of the drug in vivo fortherapeutic applications. The invention additionally provides certaindrug-polymer and drug-oligomer conjugates which are useful in thepreparation of particles for delivery of the drug in vivo. The inventionalso provides a method of making particulate drug delivery systems orvehicles employing the drug-polymer or drug-oligomer conjugates of thisinvention. The polymer and oligomer conjugates of this invention can beemployed in any art-known method for the preparation of particles frompolymers or oligomers. The methods herein are particularly useful forthe preparation of nanoparticles for drug delivery and more particularlyare useful for the preparation of nanoparticles for chemotherapyapplications. Nanoparticles of this invention are, for example, preparedby nanoprecipitation methods from the polymer and/or oligomer conjugatesdescribed in this invention.

In specific embodiments, the invention provides particles containing aselected drug optionally for sustained or targeted drug delivery. Inspecific embodiments, a selected drug is substantially (90% or more byweight of the drug) covalently bound to polymer or oligomer in theparticle. The particles are prepared, for example, by known methods fromsolutions containing drug-polymer and/or drug-oligomer conjugates. Thedrug conjugates of this invention are formed during polymerization ofthe polymer or oligomer in which the drug is employed as an initiator ofthe polymerization of the monomers which form the polymer and/oroligomer. More specifically, the drug conjugates are formed byring-opening polymerization of cyclic monomers in the presence of anappropriate ring-opening polymerization catalyst and the initiator (thedrug).

In specific embodiments, the drug-polymer or drug-oligomer conjugates ofthis invention are employed to make various types of particles usefulfor drug delivery containing the drug and ranging generally in size fromabout 2 nm to about 500 microns. In other more specific embodiments, thedrug-polymer or drug-oligomer conjugates of this invention are employedto make microparticles containing the drug and ranging generally in sizefrom about 500 nm to about 100 microns. In other embodiments, thedrug-polymer or drug-oligomer conjugates of this invention are employedto make nanoparticles containing the drug and ranging generally in sizefrom about 55 nm to about 600 nm. In other embodiments, the drug-polymeror drug-oligomer conjugates of this invention are employed to makeparticles containing the drug and ranging generally in size from about 2nm to about 100 nm. In other embodiments, the drug-polymer ordrug-oligomer conjugates of this invention are employed to makeparticles containing the drug and ranging generally in size from about200 nm to about 800 nm. In other embodiments, the drug-polymer ordrug-oligomer conjugates of this invention are employed to makeparticles containing the drug and ranging generally in size from about 1micron to about 500 micron.

In a specific embodiment, the methods of this invention can be employedto make nanoparticles in the 20-60 nm size range. Such nanoparticles canbe made, for example by known micellation methods from polymer oroligomer conjugates as described herein followed by further reactionwith a PEG-capping agent, for example PEG-isocyanate.

In another specific embodiment, the methods of this invention can beemployed to make nanoparticles in the 1-20 nm range which areparticularly useful for delivery to cells. Such nanoparticles are formedemploying cyclic AB2 type monomers or mixtures of such monomers withother cyclic esters and carbonate monomers described herein above. AB2type monomers polymerize in the methods herein to form hyperbranched ordendritic structures conjugated to a selected drug molecule. Particlesformed directly by polymerization of the AB2 type monomers can be usedfor drug delivery. Alternatively, these particles can be subjected tosurface treatments as discussed herein below.

The method of this invention is useful for forming polymer or oligomerconjugates with any small molecule drug (i.e., a small molecule drug isa non-peptide, non-sugar and non-nucleic acid-based drug) which containsat least one functional group which can function for initiation of thering-opening polymerization reaction, e.g. a hydroxyl group or a thiolgroup. The drug may contain, but need not contain, a plurality of suchpolymerization initiation groups, e.g., a plurality of hydroxyl groupsor thiol groups. In preferred embodiments, the drug contains only one ofsuch polymerization groups. The hydroxyl groups may be primary,secondary or tertiary hydroxyl groups. Similarly, the thiol groups maybe primary, secondary or tertiary thiol groups. The hydroxyl group mayalso be a phenolic hydroxyl group. In specific embodiments, the drugcontains one or more non-phenolic hydroxyl groups. In specificembodiments, the drug contains one or more non-phenolic hydroxyl groupswhich are primary or secondary hydroxyl groups. In specific embodiments,the drug contains a single non-phenolic hydroxyl group. In specificembodiments, the drug contains a single primary or secondary hydroxylgroup. Exemplary drugs which can be employed in the methods herein arelisted and illustrated in FIGS. 8 and 13 and additional drugs are listedbelow.

In specific embodiments, the drug is hydrophilic and in relatedembodiments, the drug is water-soluble (e.g., exhibiting solubility inwater in the range of mg/mL). In other specific embodiments, the drug ishydrophobic and in related embodiments, the drug is not water-soluble orexhibits low water solubility (e.g., exhibiting solubility in water inthe range of micrograms per mL or less).

In specific embodiments, the drug which is conjugated to the polymer oroligomer in the methods herein is a drug that is an anticancer agent orthat is useful in chemotherapy. In specific embodiments, the drug is ataxane. In other specific embodiments, the drug is an anticancer agentof the anthracyclin family. In other embodiments, the drug is a proteaseinhibitor. In other specific embodiments, the drug is an inhibitor ofreverse transcriptase. In other specific embodiments, the drug is anantiviral agent. In other specific embodiments, the drug is anantifungal agent. In other specific embodiments, the drug is a phenolicdrug, i.e., having one or more phenolic hydroxyl groups. In otherspecific embodiments, the drug is a thiol drug, i.e., having one or morethiol groups.

The method of this invention is also useful for delivery of drugs whichare peptides, proteins, sugars and/or nucleic acid (DNA or RNA). In eachcase, the drug must contain at least one functional group that canfunction as an initiator in the ring-opening polymerization reaction,e.g., at least one hydroxyl or one thiol group.

The method of this invention can be more broadly applied to any moleculeor other chemical species (including synthetic, or naturally-occurringmolecules and organic or inorganic species) which contains at least onefunctional group which can function as an initiator in a ring-openingpolymerization reaction (e.g., a hydroxyl group or a thiol group) andwhich one wishes to administer or deliver in vivo using a particulatedelivery system such as a microparticle or a nanoparticle. The methodmay be applied to form polymer or oligomer conjugates with any suchuseful chemical species including without limitation, reagents fordiagnostic methods, nutrients or vitamins (which may also be considereddrugs), or reporter molecules (e.g. radiolabeled or fluorescentlylabeled molecules). The chemical species to be conjugated to the polymeror oligomer may be hydrophilic, hydrophobic, water-soluble orwater-insoluble. The chemical species may contain a plurality ofhydroxyl groups or thiol groups which may be primary, secondary ortertiary hydroxyl groups and which may be phenolic hydroxyl groups. Inspecific embodiments, the hydroxyl groups are primary or secondaryhydroxyl groups. In specific embodiments, the hydroxyl groups arephenolic hydroxyl groups. In specific embodiments, the thiol groups areprimary or secondary hydroxyl groups. In specific embodiments, thechemical species is a chemical species other than a saccharide. Inspecific embodiments, the chemical species is a chemical species otherthan a carbohydrate.

In specific embodiments of all of the above-listed cyclic monomer, oneor both of Y₁ and Y₂ are haloalkyl groups, particularly fluoroalkylgroups.

The scope of the invention as described and claimed encompasses the useof racemic forms of the cyclic monomers as well as the individualenantiomers and non-racemic mixtures thereof.

The methods herein employ any appropriate ring-opening polymerizationcatalyst which may be a metal-containing catalyst or an organocatalyst.In specific embodiments, the catalysts are selected from Mg(II) orZn(II) catalysts. In other specific embodiments,1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD), N-methyl-TBD (MTBD), or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are employed asorganocatalysts.

The molar ratio of combined monomer(s) to initiator(s) ranges mostgenerally from 2:1 to 5000:1, and more specifically from 5:1 to 200:1and yet more specifically from 10:1 to 100:1. In the methods herein twoor more different cyclic monomers may be combined in the polymerizationreaction to form copolymer conjugates. In the methods herein two or moredifferent cyclic monomers may be sequentially added to thepolymerization reaction to form block co-polymer conjugates. Two or morechemical species each having at least one hydroxyl or thiol group can becombined in the methods herein to provide a mixture of polymer oroligomer conjugates with the different chemical species. For example,two different drugs each having at least one hydroxyl group or thiolgroup may be combined in the polymerization method herein to form amixture of polymer or oligomer drug conjugates. It can be beneficial,for example, to combine two or more drugs exhibiting differentmechanisms of action for treatment of the same or related disorders,diseases or conditions.

In specific embodiments, particles, particularly nanoparticles, formedby the methods herein can exhibit drug loading (or more generallyloading of the selected chemical species) that is 20% or more, 30% ormore, 40% or more, or 50% or more.

In specific embodiments, particles, particularly nanoparticles, formedby the methods herein can exhibit long circulation lifetimes useful foreffective in vivo delivery. This is particularly the case when theparticles are surface modified employing methods described herein oremploying methods that are known in the art. In specific embodiments,particles, particularly nanoparticles, formed by the methods herein canexhibit stability in salt solutions.

In the methods herein, formation of polymer or oligomer conjugates canbe combined with any known method for the formation of particles,including nanoprecipitation, micellation, emulsion and double emulsionmethods.

The invention also provides certain polymer or oligomer conjugates whichare prepared by the polymerization methods herein. These conjugates canin general be those with any chemical species that it is desired todeliver in a particulate delivery system and particularly are drugs andmost particularly are anticancer or chemotherapeutic drugs. In specificembodiments, the conjugates are those in which the polymer of theconjugate on average has 100 or fewer monomer units. In otherembodiments, the conjugates are those in which the polymer of theconjugate has on average 75, 50, or 25 monomer units. In specificembodiments, the conjugates are those in which the polymer has weightaverage molecular weight of 5000 or less, 2500 or less, 1500 or less, or1000 or less. In specific embodiments, the invention provides certainpolymer or oligomer conjugates prepared by the polymerization methodsherein and in which the chemical species of the conjugate is conjugatedor bonded to only one polymer or oligomer. In specific embodiments, theinvention provides certain polymer or oligomer conjugates prepared bythe polymerization methods herein and in which the chemical species ofthe conjugate is conjugated or bonded to only one polymer or oligomerand at only one site in the chemical species.

In specific embodiments, the invention provides polymer or oligomerconjugates to hydrophilic chemical species, and in particular tohydrophilic drugs. In other specific embodiments, a hydrophobic chemicalspecies, particularly a hydrophobic drug is conjugated to the polymer oroligomer.

The invention further provides particles, including microparticles andnanoparticles, comprising the polymer conjugates or oligomer conjugatesof this invention which are useful for in vivo delivery of selectedchemical species, more particularly one or more drugs and mostparticularly one or more anticancer or chemotherapeutic agents. Inspecific embodiments, the particles, including microparticles ornanoparticles are surface-modified by any means known in the art, forexample, with one or more antibodies, with one or more nucleic acidmolecules, e.g., aptamers, with one or more peptides or proteins, e.g.,enzymes, with one or more polymers or oligomers, e.g., amphiphilicpolymers, particularly amphiphilic polymers containing PEG.Surface-modification of particles as is known in the art can facilitatetargeting of particles to certain tissue, can facilitate entry ofparticles into cells or can enhance stability of the particle. Forexample, nanoparticles formed from polyesters, polycarbonates ormixtures thereof can be coated with hydrophilic polymers such as PEG oramphiphilic polymers containing PEG to enhance circulation lifetime ofthe nanoparticle.

In additional embodiments, the invention provides particles having acore/shell structure or having a multiple layer structure in which atleast one of the core or shell or one of the multiple layers is a layerwhich is formed from the drug (or other chemicalspecies)-polymer/oligomer conjugates of this invention. In particular,the invention relates to nanoparticles having a core/shell structures inwhich the core or shell is formed from a polymer/oligomer conjugate ofthis invention. More specifically, nanoparticles can be formed with acore that is formed from a first polymer/oligomer conjugate and a shellthat is formed from (1) a polymer, e.g., a hydrophilic polymer or anamphiphilic polymer or (2) a second polymer/oligomer conjugate of thisinvention. In specific embodiments, the first and secondpolymer/oligomer conjugates can be selected from those of a taxane, ananthracycline antibiotic, or a Shh antagonist which has a functionalgroup, such as a hydroxyl or thiol group that can function forpolymerization initiation as described herein. In more specificembodiments, the first and second polymer/oligomer conjugates can beselected from those of Ptxl, Dtxl, Doxo, cyclopamine, or camptothecin.The invention specifically provides multiple layer nanoparticlescontaining three or more different layers wherein at least one layer isformed from a polymer/oligomer conjugate of this invention.Nanoparticles include those having three, four or five layers.Nanoparticles include those in which all layers are formed frompolymer/oligomer conjugates of this invention. Nanoparticles includethose in which at least one layer is formed from a polymer/oligomerconjugate of this invention and at least one other layer is formed froma polymer (non-conjugated polymer) such as a hydrophilic, hydrophobic oramphiphilic polymer. In specific embodiments, nanoparticles includethose in which at least one layer is formed from a polymer/oligomerconjugate of this invention and at least one other layer is formed froman amphiphilic polymer comprising PEG.

The invention additionally provides methods for making a medicamentemploying the polymer or oligomer conjugates of this invention as wellas the medicaments made thereby. Medicaments are particles, particularlynanoparticles, formed from the conjugates of this invention.

The invention further provides kits for carrying out the polymerizationreactions herein to form polymer or oligomer conjugates with a selectedchemical species having at least one hydroxyl group. The kits compriseone or more containers which in turn comprise one or more cyclicmonomers and one or more ring-opening polymerization catalysts andoptionally include instructions for carrying out the polymerizationreaction, instructions for making particles, one or more reagents orinstructions for surface modification of particles, one or more solventsfor carrying out the polymerization or for making particles, one or morecontrol initiators, additional receptacles for carrying out thereaction, for forming particles or for carrying out surfacemodification. In specific embodiments, kits herein comprise a pluralityof different cyclic monomers useful for making conjugates with differentoligomers or polymers. In other embodiments, kits herein can furthercontain one or more different chemical species having at least onehydroxyl group for forming conjugates.

Additional embodiments of the invention will be apparent on review ofthe following detailed description, examples and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic illustrations of the formation ofnanoencapsulates (prior art) and nanoconjugates of this invention,respectively. The figures illustrate the structural differences betweenthe nanoencapsulates and the nanoconjugates.

FIG. 2A is a schematic illustration of lactide polymerization of theinvention exemplified for drug initiation of polymerization in thepresence of catalyst for the preparation of high loading, 100%incorporation efficiency, controlled-releasing nanoconjugates for invivo application.

FIG. 2B provides a specific example of synthesis of exemplifiedpegylated pacitaxel-containing nanoconjugates by Ptxl initiatedpolymerization of lactide in the presence of (BDI)MN(TMS)₂ (where M=Mgor Zn), followed by nanoprecipitation and non-covalent surfacepegylation with PLGA-mPEG. The polymerization is believed to beinitiated through formation of a (BDI)M-oxide with Ptxl.

FIG. 3 is a graph of the release kinetics at 37 C in 1×PBS of Pxtl fromPxtl-LA nanoconjugates: Pxtl-LA₂₅ NC and Pxtl-LA₅₀ NC (drug loadingindicated in figure). Also included in the graph for comparison is therelease kinetics of a Pxtl/PLA nanoencapsulate (NE) prepared bynanoprecipitating a mixture of Ptxl and PLA (Pxtl/PLA (wt/wt)=1/12).

FIG. 4 is a graph showing toxicity evaluation of Ptxl-LA₅₀ NC, Ptxl-LA₂₅NC, Ptxl-LA₁₀ NC and Pxtl using the MTT assay in PC-3 cells after 24 hincubation. The “*” indicates significance at 95% confidence interval.

FIG. 5 is a graph showing changes in particle size when PLGA-mPEG_(5k)is added to Pxt-LA₂₀₀ NC. Particle size increases linearly as a functionof the weight ratio of amphiphilic copolymer to drug-polymer conjugate.

FIG. 6 is a graph illustrating the stability of Ptxl-LA₂₀₀ NC in PBS at37 C before (▪) and after treatment with PLGA-mPEG_(5k) (♦) or mPEG_(5k)().

FIGS. 7A-D are graphs showing the results of MTT studies of Ptxl-LA,Dtxl-LA, CPT-LA and Doxo-LA NCs cytotoxicity on PC-3 prostate cancercells.

FIG. 8 provides the chemical formulas for several drugs or otherchemical species (e.g., Cy5 reported dye) which have been incorporatedinto NCs employing the methods herein.

FIG. 9 is a schematic illustration of a method for preparation ofdendritic nanoparticles, which are believed to be unimolecular dendriticparticles containing one therapeutic in each nanoparticle. Thepolymerization method is similar to that described. These dendriticparticles give <20 nm particle size, a range unachievable using otherstrategy.

FIG. 10A is a graph illustrating the change in size of nanoparticles(nm) during a multidrug layer-by-layer precipitation process. In thisprocess, a nanoconjugate formed by nanoprecipitation of a drug-polymerconjugate of this invention is treated with a second drug-polymerconjugate, under nanoprecipitation conditions, to form a shell or secondlayer of the second drug polymer in the nanoconjugates. The figureprovides a size distribution plot of nanoparticle size change on theformation of a nanoparticle with a Dtxl-LA₁₀₀ core and a Doxo-LA₁₀₀shell or second layer.

FIG. 10 B is a plot of nanoparticle size (nm) as a function of theamount of shell-forming (second drug-polymer) conjugate onnanoprecipitation of a second drug polymer conjugate onto nanoparticles(NCs) formed from a first drug polymer conjugate. The plot compares sizechange as a function of the amount of the second drug-polymer conjugateadded. In one case, the second drug conjugate is the same as the firstdrug conjugate and the plot illustrates the effect of adding increasingamounts of the same drug-polymer conjugate.

FIGS. 11A and 11B provide the results of luciferase assays of NCcontaining cylcopamine added to Shh-Light 2 cells bearingluciferase-encoded Gli-1gene. FIG. 11A shows results with addition ofCA-LA₁₀ and CA-LA₂₅ where loading is indicated in parentheses. Thefigure shows that the EC₅₀ of the cyclopamine NCs are significantlylower than that of free cyclopamine, demonstrating that thenanoconjugate allowed delivery, concentrated accumulation and release ofcyclopamine in the targeted cells. FIG. 11B shows the results ofculturing the Shh-Light 2 cells with CA NCs along with NEs containingpurmorphamine which is a Shh-agonist.

FIG. 12 provides the actual MW and PDI of Ptxl-LA conjugates, measuredby GPC, formed using different catalysts (shown in the figure) in the LApolymerization synthesis of the conjugates.

FIG. 13 is a partial list of drugs that are useful in the methods of theinvention. The list includes structures.

DETAILED DESCRIPTION OF INVENTION

The present invention is based at least in part on the discovery thatpolymer and oligomer conjugates with drugs and other chemical speciesthat can function as an initiator of ring-opening polymerization can bereadily prepared in a single step polymerization synthesis in which thechemical species initiator is combined with one or more cyclic monomersand a ring-opening polymerization catalyst. FIG. 2 schematicallyillustrates the methods of this invention (A) and illustrates a specificexample of nanoconjugate formation using paclitaxel conjugated to PLA.Further, it has been discovered that the polymer and oligomer conjugatesthus formed are useful in the preparation of particles, includingmicroparticles and nanoparticles, having particle sizes that are usefulfor the delivery of the chemical species in vivo. As specificallyillustrated in FIG. 2, nanoprecipitation methods can be used to formnanoparticles (nanoconjugates) containing the conjugates of thisinvention. As further illustrated in FIG. 2 nanoparticle nanoconjugatescan be treated with PEG to peglyate the surface of the nanoparticle.

Polymer and oligomer conjugates formed by the methods herein aredistinguishable from conjugates formed by conjugation of a chemicalspecies, particularly a drug, with a pre-formed polymer. The conjugatesformed can exhibit polymer average molecular weight much lower thanpre-formed polymers. The conjugates formed can exhibit polydispersitymust lower than pre-formed polymers. For example, polymer conjugates ofthis invention can exhibit polydispersities of 1.5 or less, 1.3 or lessand 1.2 or less. In general the conjugates formed by the methods hereinwill be more uniform in polymer length than those formed by conjugationof a chemical species with a pre-formed polymer.

Nanoconjugates (NCs) formed from nanoprecipitation of the polymer oroligomer conjugates of this invention are distinguishable fromnanoencapsulates (NEs) with respect to drug loading, drug encapsulation,drug release, particle distribution as well as ease of manufacture, asillustrated in FIG. 1. NEs exhibit low to medium drug loading (1-5 wt%), which it is not possible to predetermine and which can vary frombatch to batch. NCs exhibit predefined drug loading levels with muchhigher batch to batch consistency. NEs exhibit uncontrollableencapsulation efficiency (ranging 10-80%), which vary frombatch-to-batch, and system-to-system and which are unable to encapsulatehydrophilic drugs. NC's exhibit circa 100% encapsulation efficiency,with little or no batch-to-batch and system-to-system variation and canbe formed with both hydrophilic and hydrophobic drugs. NEs exhibitsignificant burst release with 40-80% release in the first 24 hours. NCsexhibit little or no burst release of drug and provide for adjustableand controllable release of drug. NEs usually exhibit multimodalparticle distributions. NCs exhibit monomodal particle distribution. Themanufacture of NEs involves a multi-component/multi-step process whichis difficult to scale up and detrimental for long-term storage. Furtherit is difficult to remove unencapsulated drug and requires difficult touse filtration method for sterilization. In contrast, the manufacture ofNC involves a single component system which is straightforward to scaleup and when properly stored, drug release should be minimal increasingstorage lifetime. Because there is essentially no free drug or drugaggregate to remove, the method is simpler and less costly to implement.

During polymerization as illustrated in FIG. 2 part A, the chemicalspecies that functions for polymerization initiation (e.g., drug)becomes covalently bonded to one or more growing oligomer or polymerchains. The polymerization reaction is a ring-opening polymerizationreaction which preferably has the characteristics of a livingpolymerization. The invention has been exemplified herein with drugs andother chemical species having one or more hydroxyl groups or thiolgroups which can function in the presence of certain catalysts aspolymerization initiators. As discussed herein the ring-openingpolymerization can employ various cyclic monomers, including cyclicesters, cyclic carbonates as well as cyclic siloxanes and cyclicphosphorous containing monomers. The polymerization can be exemplifiedfor the polymerization of a lactide or glycolide and with a chemicalspecies which is a drug and which carries one or more hydroxyl or thiolgroups.

Numerous alcohol-metal oxides (RO-M) have been developed for controlled,living polymerization of lactide and other related cyclic monomers withquantitative, terminal conjugation of RO to polylactide through an esterbond.¹⁶ The amount of RO in the resulting polylactide can be preciselycontrolled by adjusting lactide/ROH ratio (e.g., the monomer toinitiator molar ratio). In the present invention, ROH is a drug or otherchemical species containing one or more hydroxyl groups that are to beconjugated to the polymer formed on ring-opening polymerization. Anumber of organocatalysts, such as TBD(1,5,7-Triazabicyclo[4.4.0]dec-5-ene) can also be employed with hydroxylor thiol containing initiators (i.e., drugs or other species to beconjugated) to form the conjugates of this invention. In these cases aswell, the amount of the drug or other chemical species in the resultingpolymer (or oligomer) is controlled by controlling the monomer/initiatorratio. Additional exemplary catalysts are provided in Example 6. Theeffect of varying catalyst on the actual MW of durg-polymer conjugatesis illustrated in FIG. 12.

As part of this work, it was demonstrated that hydroxyl-containingchemotherapeutics could be quantitatively incorporated into polylactideusing this polymerization method (exemplified with Ptxl in FIG. 2 part Busing a Mg(II) complex ((BDI)MgN(TMS)₂ to activate Ptxl). As aconsequence, drug release rate from the particle can be modulated by thecleavage of drug-polylactide ester bond, which is much more controllablethan the diffusion of the encapsulated non-covalently bonded drug from aparticle. Release kinetics of the drug would then be controlled byadjusting drug loading and particle size. Because polymerizationreactions can be controlled to give quantitative yield, drug loading canin turn be precisely controlled simply by adjusting monomer/drug (orother species) molar ratio (monomer to initiator ratio). The capabilityof the present methods to precisely control drug loading by controllingthe drug-polymer composition will significantly enhance clinicaltranslation of the nanoparticle products and the likelihood forregulatory approval of the nanoparticles for clinic use. In addition,unprecedented high drug loading have been demonstrated (up to ˜40%) withnanoparticles generated by the methods of this invention.

Nanoparticles can be formed from polymer and/or oligomer conjugates ofthis invention by various known methods. In a specific embodiment,nanoprecipitation is employed in which a solution of the conjugate isadded to a solution in which the conjugate is insoluble. Theprecipitation step for forming nanoparticles employing the conjugates ofthis invention is simplified compared to the use of other startingmaterials because only one type of material, the conjugate is involved.The precipitation and encapsulation of a free drug in a polymer, even ina binary system, in contrast can be very complex. Control of integrationof drug and polymer during phase separation is often poor especiallywhen these two elements have distinct chemical and physical properties.Biphasic particle distributions have been consistently observed innanoencapsulates which may be due in part to the self-aggregation ofdrug or polymer according to the like-dissolves-like principle. Themethod of this invention provides particles with monomodal particledistributions.

The benefits described above for drug-polymer or drug-oligomerconjugates will generally be observed in the formation of conjugateswith any chemical species which can function as polymerizationinitiators and which it is desired to delivery in vivo in particulateform. Further, the specific benefits described above for the preparationof nanoparticle delivery compositions will generally be observed whenthe polymer or oligomer conjugates are employed to make any sizeparticle that is useful for in vivo delivery.

Among various NP preparation methods, nanoprecipitation has been widelyused for preparing NPs for use as encapsulated chemotherapy drugs. In atypical approach, a degradable hydrophobic polymer, such as polylactide,is mixed with a hydrophobic drug in a water-miscible solvent (e.g. THFor DMF) and added to excess water. Diffusion of the organic solvent intowater facilitates the formation of sub-100 nm sized nano-aggregates withrandomly mixed drug and polymer molecules. More generally any of themany methods that are known in the art for preparing nanoparticles frompolymer or oligomeric materials can be employed with the conjugates ofthis invention.

In one aspect, the invention relates to a nanoconjugation techniquewhich integrates drug-initiated cyclic ester (or carbonate)polymerization and nanoprecipitation to prepare drug-containingnanoparticles with pre-defined drug loading, near 100% encapsulationefficiency, minimized particle heterogeneity and significantly reducedburst release effect. In applications for cancer chemotherapy andparticularly with nanoparticles useful in such therapy, particulateformulations of this invention will exhibit improved efficacy anddecreased toxicity.

In an embodiment the invention relates to polymeric nanoparticles forcancer treatment. In this aspect of the invention, the drug-polymer ordrug-oligomer conjugate includes an anticancer agent or chemotherapeuticagent. Nanoparticles useful for cancer treatment may contain a mixtureof conjugates with two or more anticancer agents.

Particulate formulations (i.e., those containing NP and NC) of thisinvention can be administered to a subject by any known methodappropriate for the size of the particle and the therapeutic, diagnosticor other agent carried in the particulate.

This invention additionally relates to the use of drug-conjugates withpolymers and oligomers in the preparation of a medicament for in vivodelivery of the drug. The drug can, for example, be an anticancer agent.More specifically, the invention relates to the use of a drug in themanufacture of a medicament for treatment of cancer. In specificembodiments the medicament manufactured is in the form of particles,particularly nanoparticles, for administration in any appropriate dosageform. In specific embodiments, the medicament further comprises apharmaceutically acceptable carrier or diluent and particularly acarrier or diluent suitable for the desired form of administration.

The term drug is employed herein very generically to include anychemical species that can provide therapeutic benefit to an individualin need of such benefit. Conjugates here can be formed with appropriatedrugs or other chemical species which one wishes to deliver in ananoparticle. The drug or other chemical species must have at least onefunctional group that can function in the presence of a catalyst forinitiation of ring-opening polymerization. It is believed that thefunctional group must be capable of interaction with the catalyst toform a species active for polymerization. Hydroxyl groups and thiolgroups, for example, are capable of functioning for initiation ofring-opening polymerization. Hydroxyl and thiol groups can be primary,secondary or tertiary functional groups. As is understood in the art,primary, secondary and tertiary hydroxyl and thiol group have differentsteric environments and can exhibit different relative reactivities.

In the description of chemical groups herein the terms used are intendedto have their broadest art-recognized meaning.

The chemical species having at least one functional group functional forinitiation of ring-opening polymerization is combined with one or morecyclic monomers which can be polymerized by ring-opening polymerizationand an appropriate ring-opening polymerization catalyst in anappropriate solvent under conditions and for a sufficient time to formoligomers or polymers as desired. A variety of chemical species areknown to function for initiation of ring-opening polymerization in thepresence of appropriate catalysts. Among these chemical species arethose which contain one or more hydroxyl groups or one or more thiolgroups in their chemical structure. The ability of a given chemicalspecies to function for polymerization initiation as required for thisinvention can be readily assessed without undue experimentation in testpolymerization reactions carried out employing materials and methods astaught herein or as well-known in the art. The methods of the inventionhave been, for example carried out successfully with drug and otherspecies illustrated in FIG. 8. FIG. 13 contains a number of drugscontaining hydroxyl groups that are useful in the preparation ofdrug-conjugates and nanoconjugate particles of this invention.

Additional drugs carrying hydroxyl groups which are useful in themethods of this invention include, among others, Darunavir (TMC-114),Tipranavir (TPV), Saquinavir (SQV), Ritonavir (RTV), Indinavir,Nelfinavir (NFV), Amprenavir (APV), Lopinavir (ABT-378), Atazanavir(ATV), Vinorelbine bitartrate, fulvestrant, Sarcodictyins,camptothecins, Vinblastine, bryostatin 1, (+)-Cylindricine,(+)-Lactacystin, Aeruginosin 298-A, (+)-Fostriecin, GarsubellinA/Hyperforin, (S)-Oxybutynin, Epothilone A, Zidovudine (AZT), Lamivudine(3TC), Didanosine (ddI), Abacavir (ABC), and Emtricitabine (FTC)

Additional drugs useful in the methods of this invention include thoseof various structures, but which have phenolic hydroxyl groups, whichinclude among others include, bamethane, ethamivan, hexachlorophene,salicylanilide, pyrocatechin, thymol, pentazocine, phloroglucinol,eugenol, niclosamide, terbutaline, dopamine, methyldopa, norepinephrine,eugenol, α-naphthol, polybasic phenols, adrenaline, dopamine,phenylephrine, metaraminol, fenoterol, bithionol, alpha-tocopherol,isoprenaline, adrenaline, norepiniphrine, salbutamol, fenoterol,bithionol, chlorogenic acid/esters, captopril, amoxicillin, betaxolol,masoprocol, genistein, daidzein, daidzin, acetylglycitin, equol,glycitein, iodoresiniferatoxin, SB202190, and tyrphostin SU1498.

For a given chemical species that it is desired to conjugate by themethod herein, it may be necessary to perform trial polymerizationsemploying different catalysts, for example, certain chemical specieswill be more compatible with organometallic catalysts, while others maybe more compatible with organocatalysts. For example, it has been foundthat the chemical species even though containing appropriate functionalgroups may not function (or may have limited function) to initiatepolymerization with certain metal-based catalysts because the chemicalspecies may deactivate the catalysts. Specifically, conjugation of PLAwith mitoxantrone employing (BDI)MgN(TMS)₂ did not proceed. It isbelieved that the mitoxantrone (Formula J) which has hydroxyalkyl aminegroups may have deactivated the Mg catalyst.

Any of the cyclic monomers described herein including AB2 type cyclicmonomers can be employed to form polymer or oligomer conjugates withsuch chemical species that can function for polymerization initiation.Any cyclic monomer or mixtures thereof that can be polymerized byring-opening polymerization can be employed to form the drug-conjugatesand particles, particularly nanoparticles, of this invention. Inparticular, cyclic monomers that can be polymerized by activated —OH ora metal-oxide group can in general be employed to form thedrug-conjugates and particles of this invention. Useful cyclic monomersinclude cyclic esters and cyclic carbonates. Cyclic esters include,lactones, cyclic diesters, and cyclic ester-amides, e.g., cyclicdepsipeptides.

Cyclic esters have the formula:

where m+n ranges from 1-20, X is O or NH, x is 0 or 1 to indicate thepresence of the ester or amide group and Y₁ and Y₂ indicate the optionalsubstitution of one or more carbon atoms of the ring with non-hydrogensubstituents. Each Y₁ and Y₂, independently of one another aresubstituents that do not interfere with the polymerization reactions asdescribed herein and can for example be selected from the groupconsisting of hydrogen, halogen, —COOR, —NRR′, —SR, —OR, where R and R′independently are one or more hydrogens, alkyl or aryl groups, aguanidinium group, an imidiazole group, an alkyl group, alkenyl group,alkynyl group, aryl group (including phenyl or benzyl) and —N₃. Each Y₁or Y₂ can also be an amino acid or short peptide having 1-5 amino acids.Each Y₁ or Y₂ also include groups as listed above which are protectedwith an art-recognized protecting group. Alkyl, alkenyl, alkynyl andaryl groups are optionally substituted with one or more halogens(including one or more fluorines), —N₃, —COOR″, —NR″R′″, —SR″, —OR″where R″ and R′″ are independently hydrogen or an unsubstituted alkyl,alkenyl, alkynyl or aryl group. In a specific embodiment, one or two ofY₁ and Y₂ can be a hydroxyl alkyl group. In specific embodiments, eachY₁ and Y₂ is a hydrogen or an alkyl group having from 1 to 6 carbonatoms, particularly a methyl group.

Cyclic carbonates have the formula:

where p ranges from 1-20 and Y₁ and Y₂ indicate the optionalsubstitution of one or more carbon atoms of the ring with non-hydrogensubstituents and where each Y₁ and Y₂ are as defined above. In specificembodiments, each Y₁ and Y₂ is a hydrogen or an alkyl group having from1 to 6 carbon atoms, particularly a methyl group. In a specificembodiment, one or two of Y₁ and Y₂ can be a hydroxyl alkyl group.

Cyclic esters include, without limitation, lactones such asβ-butyrolactone (n=2), δ-valerolactone (n=4), ε-caprolactone (n=5),α-methyl-β-propriolactone, β-methyl-β-propriolactone,ω-pentadecalactone, ω-dodecalactone and any lactide or glycolideincluding all stereo-isomers thereof, e.g.

any substituted lactide or glycolide:

any cyclic depsipeptides (half-ester and half-amide) with 6 or 7 memberring structure, including, among others, Formulas D1-D3:

respectively.

Other cyclic monomers that are polymerizable by activated —OH ormetal-oxide group, include phosphorus-containing cyclic esters includingcyclic phosphates and phosphonates:

where q=1 to 20, Y₃ is as defined for Y₁ and Y₂ above and R₃ is Y₃(phosphonates) or —OY₃ (phosphates).

Cyclic Phosphonites:

where variables are as defined above,and silicon-containing cyclic monomers including

where each R is independently selected from hydrogen or an optionallysubstituted alkyl group. In specific embodiments of the above cyclicmonomers, each of Y₁₋₃ are hydrogen or alkyl groups having 1-6 carbonatoms. In specific embodiments of the above cyclic monomers, all Y₁₋₃are hydrogen or all Y₁₋₃ are alkyl groups having 1-6 carbon atoms,particularly all Y₁₋₃ are methyl groups. In specific embodiments of theabove cyclic monomers, each R is selected from hydrogen or an alkylgroup having 1-6 carbon atoms. In specific embodiments of the abovecyclic monomers, all R's are hydrogen or all R's are alkyl groups having1-6 carbon atoms, particularly all R's are methyl groups.

In specific embodiments, AB2 type cyclic polymerizable monomers areemployed alone or in combination with other cyclic esters or cycliccarbonates. AB2 type cyclic ester monomers include those of formula:

where z is 1 to 6 and Y₁ is as defined above. In specific embodiments,Y₁ can be hydrogen or an alkyl group having from 1-6 carbon atoms.

The term “alkyl” refers to a monoradical of a branched or unbranched(straight-chain or linear) saturated hydrocarbon and to cycloalkylgroups having one or more rings. Unless otherwise indicated preferredalkyl groups have 1 to 20 carbon atoms and more preferred are those thatcontain 1-10 carbon atoms. Short alkyl groups are those having 1 to 6carbon atoms including methyl, ethyl, propyl, butyl, pentyl and hexylgroups, including all isomers thereof. Long alkyl groups are thosehaving 8-20 carbon atoms and preferably those having 12-20 carbon atomsas well as those having 12-20 and those having 16-18 carbon atoms. Theterm “cycloalkyl” refers to cyclic alkyl groups having preferably 3 to20 carbon atoms having a single cyclic ring or multiple condensed rings.Cycloalkyl groups include, by way of example, single ring structuressuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl,and the like, or multiple ring structures such as adamantanyl, and thelike. Unless otherwise indicated alkyl groups including cycloalkylgroups are optionally substituted as defined below.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group having one or more double bonds and tocycloalkenyl group having one or more rings wherein at least one ringcontains a double bond. Unless otherwise indicated preferred alkylgroups have 1 to 20 carbon atoms and more preferred are those thatcontain 1-10 carbon atoms. Alkenyl groups may contain one or more doublebonds (C═C) which may be conjugated or unconjugated. Preferred alkenylgroups are those having 1 or 2 double bonds and include omega-alkenylgroups. Short alkenyl groups are those having 2 to 6 carbon atomsincluding ethylene (vinyl), propylene, butylene, pentylene and hexylenegroups including all isomers thereof. Long alkenyl groups are thosehaving 8-20 carbon atoms and preferably those having 12-20 carbon atomsas well as those having 12-20 carbon atoms and those having 16-18 carbonatoms. The term “cycloalkenyl” refers to cyclic alkenyl groups of from 3to 20 carbon atoms having a single cyclic ring or multiple condensedrings in which at least one ring contains a double bond (C═C).Cycloalkenyl groups include, by way of example, single ring structures(monocyclic) such as cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cyclooctenyl, cylcooctadienyl and cyclooctatrienyl as wellas multiple ring structures. Unless otherwise indicated alkyl groupsincluding cycloalkyl groups are optionally substituted as defined below.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbonhaving one or more triple bonds (C≡C). Unless otherwise indicatedpreferred alkyl groups have 1 to 20 carbon atoms and more preferred arethose that contain 1-10 carbon atoms. Alkynyl groups include ethynyl,propargyl, and the like. Short alkynyl groups are those having 2 to 6carbon atoms, including all isomers thereof. Long alkynyl groups arethose having 8-20 carbon atoms and preferably those having 12-20 carbonatoms as well as those having 12-16 carbon atoms and those having 16-18carbon atoms. The term “cycloalkynyl” refers to cyclic alkynyl groups offrom 3 to 20 carbon atoms having a single cyclic ring or multiplecondensed rings in which at least one ring contains a triple bond (C≡C).Unless otherwise indicated alkyl groups including cycloalkyl groups areoptionally substituted as defined below.

The term “aryl” refers to a monoradical containing at least one aromaticring. The radical is formally derived by removing a H from a ringcarbon. Aryl groups contain one or more rings at least one of which isaromatic. Rings of aryl groups may be linked by a single bond or alinker group or may be fused. Exemplary aryl groups include phenyl,biphenyl and naphthyl groups. Aryl groups include those having from 6 to30 carbon atoms and those containing 6-12 carbon atoms. Unless otherwisenoted aryl groups are optionally substituted as described herein. Theterm aryl includes “arylalkyl” groups which refers to a group thatcontains at least one alkyl group and at least one aryl group, the arylgroup may be substituted on the alkyl group (e.g., benzyl, —CH₂—C₆H₅) orthe alkyl group may be substituted on the aryl group (e.g., tolyl,—C₆—H₄—CH₃). Unless otherwise noted either the alkyl or the aryl portionof the arylalkyl group can be substituted as described herein.

The polymerization reaction to form conjugates of the invention can becarried out under various reaction conditions (temperature, solvent,concentrations) as is understood in the art. These conditions are inpart selected to retain activity of any chemical species, particularly adrug, that is to be conjugated. The polymerization reaction can becarried out in any appropriate solvent or mixture of solvents. In aspecific embodiment, the solvent is an anhydrous, water-misciblesolvent. The polymerization can be carried out in the same or adifferent solvent than that which is used in the later preparation ofparticles. Useful solvents for the polymerization reaction include,among others, THF, acetone, methylene chloride, chloroform,dimethylformamide, DMSO, acetonitrile or mixtures thereof.

The term polydispersity is used herein to refer to the distribution ofmolecular weights of polymers in a given sample. The PolydispersityIndex (PDI) is a specific measure of polydispersity and is the weightaverage molecular weight divided by the number average molecular weightand relates to the distribution of individual molecular weights in agiven sample of polymers. PDI can be determined using Gel PermeationChromatography (GPC). As the polymer chains in a given sample approachuniform chain length, PDI approaches 1.

Particles of this invention can be surface-modified as is known in theart to improve their usefulness as drug delivery vehicles.

For particles, particularly nanoparticles, that can successfully carrydrug molecules or other chemical species to a desired in vivo location,e.g., a tumor site and get into cancer cells, it is preferably towell-control their features so that the they can circumvent variousphysiology barriers to reach tumor tissues. Systemically administerednanoparticles without proper modification are usually cleared rapidlyfrom the circulation and localized predominately in liver and spleen.Severe liver and spleen retention not only greatly diminishes theaccessibility of the nanoparticles to target tissue, e.g., tumor tissue,but also causes liver and spleen damage. Clearance is due to thescavenging by liver Kupffer cells and spleen macrophages. Nanoparticlescan be cleared within a few to tens of minutes by this passive andsite-specific mechanism. In addition, nanoparticle surfacecharacteristics and sizes play an important role in the bloodopsonization, a process of the deposition of opsonins, like fibronectin,which will trigger immune responses and accelerate the clearance ofnanoparticles from blood by macrophages. The binding of opsonins to thesurface of nanoparticles can be substantially reduced when surfacefeatures of the nanoparticles are well controlled.

For example, nanoparticle surface pegylation, a well-establishedapproach to reduce protein binding, forms a hydrophilic layer that cansubstantially reduce blood protein binding and reduce liver and spleenuptake. Pegylation creates stealth-like structures resembling thestrategies developed by pathogenic microorganisms to bypass immunedetection. Suppression of opsonization is thus achievable and has beenutilized to enhance passive retention of nanoparticles in circulationand avoid trapping of nanoparticles in macrophages when they are incontact with blood. This simple strategy for manipulation of thenanoparticle surface can have a significant impact, as the circulationhalf-life of a nanoparticle can be increased from several minutes toseveral or tens of hours on pegylation. The use of surface pegylationhas become a very popular approach to reduce recognition by macrophagecells.

Besides surface morphology, nanoparticle size is another importantparameter that can significantly affect the biodistribution and in vivoefficacy. Nanoparticle sizes can dramatically affect the clearance rate.Large particles with size 200 nm or above are more likely to inducemacrophage immune response and activate the uptake by Kupffer cells thantheir smaller counterparts. The size of fenestrae in the sinusendothelium in liver can be as large as 150 nm. Splenic filtration atinterendothelial cell slits can predominate when particles size exceedsthat of the cell slits (200-250 nm). Therefore nanoparticle sizes areusually controlled to 150 nm or below when they are to be used inanticancer drug delivery in order to have prolonged circulation.However, the nanoparticle size should not be too small, otherwise theparticles can be very quickly filtered through the kidney (size <10 nm)which is a typical problem of polymer-drug conjugates with molecularweight of 40 kDa or lower. Very small particles (1-20 nm) can alsoslowly extravasate from the vasculature into the interstitial spaces,and are further accumulated in lymph nodes via lymphatic vessels.Nanoparticles with size smaller than 20 nm can readily escape from thevasculature into blood capillaries with open fenestration. Thereforenanoparticles must be large enough to prevent undesirable leakage fromcirculation, but must be small enough to minimize immune responses.

Most nanoparticles used for anticancer delivery are in the range of 20to 150 nm. To improve anticancer delivery, attention has been focused onthe development of stealth technologies to provide means for increasedextravasation of long circulating NPs at leaky tumor vasculature. Thevasculature of tumors is highly heterogeneous. Depending on the specificlocation, tumor tissue can be vascularly necrotic or extremelyvascularized so that adequate nutrient and oxygen can be transported tothe tumor tissue to support its fast growth. Tumor blood vessels arealso very heterogeneous and have several abnormalities when compared tonormal blood vessels. In general, tumor blood vessels are leakier thantheir normal counterparts, and are shown to have a characteristic porecutoff size ranging between 380 and 780 nm. These pores become thepathway for NPs to leave the circulation system and enter the tumorinterstitial space. Therefore NP with size of 150 nm or lower can freelydiffuse through these leaky vessel pores, while particles with sizeslarger than 400 nm are much less likely to extravasate into tumor issue.Because of the undeveloped lymphatic drainage system in tumor tissue,nanoparticles which extravasate the leaky pore of tumor vasculaturecannot be readily removed. Therefore sustained circulation results inincreased accumulation of nanoparticles over the time. This effect isthe extremely well-known Enhanced Permeation and Retention (EPR) effectpassive targeting mechanism in caner drug delivery.

The polymer and oligomer conjugates of this invention can be chemicallymodified by reaction to introduced desired terminal functional groups.Terminal functional groups of interest for applications to drug deliveryinclude among others, hydroxyl, thiol, amine, azide, alkyne, alkene,ketone, phenol, halide, imidazole, guanidinium, carboxylate, orphosphate groups. These desired functional groups can be introduced atthe terminus of the polymers or oligomers herein employing well knownchemical methods. These functional groups can be employed to furtherconjugate the polymer or oligomer conjugate of this invention with otherchemical species, such as other polymers, other oligomers,carbohydrates, peptides, proteins, antibodies, nucleic acids, aptamersetc. and/or to provide sites for surface modification for nanoparticlesprepared using the conjugates of this invention.

The invention also relates to multiple layer particles in which aparticle prepared by the methods herein is treated to coat or otherwiseprovide a second layer of polymer on the nanoparticle. The secondpolymer may be the same of different from that of the polymer of thepolymer conjugate in the particle. Particles of this invention maycontain two or more conjugated chemical species, e.g., two or moredifferent drugs, that are compatible in a given application. Particlesof the invention may contain different layers or portions in which theconcentration of the chemical species or drug is different. For examplean outer layer may contain a higher or lower concentration of a givenchemical species (e.g., drug) compared to an inner layer. For example,an outer layer may contain PEG while an inner layer contains a conjugateof a different polymer. For example, a first inner layer can contain apolymer or oligomer conjugate of a first drug, and a second outer layercontaining a polymer or oligomer conjugate of a second drug.

Nanoparticles of the invention can have a core/shell structure or have amultiple layer structure in which at least one of the core or shell orone of the multiple layers is a layer which is formed from the drug (orother chemical species)-polymer/oligomer conjugates of this invention.For example, as illustrated in examples herein the core of ananoparticle can be formed form a polymer/oligomer conjugate of thisinvention by methods described above for forming nanoparticles.Thereafter a shell can be added to the core nanoparticle to generate acore/shell nanoparticle having increased particle size. Morespecifically, a core/shell nanoparticle can be formed with a core thatis formed from a first polymer/oligomer conjugate and a shell that isformed from a polymer, e.g., a hydrophilic polymer or an amphiphilicpolymer. In specific embodiments the polymer is an amphiphilic blockco-polymer. In specific embodiments, the polymer is a polymer that is aPEG or which comprises a PEG (as the polymer or as a block of a thepolymer). Alternatively, a core/shell nanoparticle can be formed from afirst polymer/oligomer conjugate of this invention (to form the core)and a second polymer/oligomer conjugate of this invention to form theshell. Note that in a specific embodiment, one of the first or secondpolymer conjugates can be one in which a label or reporter molecule isconjugated to the polymer or oligomer. In specific embodiments, thefirst and/or second polymer/oligomer conjugates can be selected fromthose of a taxane, an anthracycline antibiotic, or a Shh antagonistwhich has a functional group, such as a hydroxyl or thiol group that canfunction for polymerization initiation as described herein. In morespecific embodiments, the first and/or second polymer/oligomerconjugates can be selected from those of Ptxl, Dtxl, Doxo, cyclopamine,or camptothecin.

Nanoparticles of the invention can be multiple layer nanoparticlescontaining three or more different layers wherein at least one layer isformed from a polymer/oligomer conjugate of this invention, includingthose of drugs or other chemical species, such as labels or reportermolecules. Nanoparticles include those having three, four or fivelayers. Nanoparticles include those in which all layers are formed frompolymer/oligomer conjugates of this invention. Nanoparticles includethose in which at least one layer is formed from a polymer/oligomerconjugate of this invention and at least one other layer is formed froma polymer (non-conjugated polymer) such as a hydrophilic, hydrophobic oramphiphilic polymer. In specific embodiments, nanoparticles includethose in which at least one layer is formed from a polymer/oligomerconjugate of this invention and at least one other layer is formed froman amphiphilic polymer comprising PEG.

Polymers comprising PEG include among others amphiphilic copolymerscomprising PEG such as poly(lactide)-PEG (PLA-PEG) an amphiphiliccopolymer that has a PLA and PEG segment,poly(glycolide-co-lactide)-b-methoxylated PEG (PLGA-mPEG), anamphiphilic copolymer that has a PLGA and PEG segment. In suchcopolymers, PEG can, for example, range from 10% to 90%, from 20% to50%, from 60% to 80%, from 50% to 75%, from 70% to 99% or from 1% to 50%of the copolymer.

Polymers and oliogmers used in the methods and materials herein arepreferably biocompatible and biodegradable (dependent upon the desiredapplication). They preferably exhibit little or no undesired toxicity inuse.

The particles of this invention can be surface-modified for preferentialtargeting to certain cell types. Preferential targeting of particles canfor example be achieved by covalent or non-covalent attachment oftargeting ligands to the surface of the particle.

The term particle is used herein generally to refer to a particle havingany given shape that has a size that is useful for in vivo delivery bysome administration method. The particles may be micelles, aggregates,sphere or have no regular shape. The term particle size is used hereinas it is generally used in the art and is determined by methodsdescribed in the Examples herein.

The invention relates to conjugates of polymers or oligomers. Mostgenerally a polymer is a chemical species containing a plurality ofrepeating units which are bonded to each other. A polymer may containmore than one different repeating unit. The repeating unit typicallyderives from polymerization of a monomer. A copolymer specificallyrefers to a polymer containing two or more structurally differentrepeating units. The different repeating units of a polymer may berandomly ordered in the polymer chain or the same repeating units may begrouped into contiguous blocks in the polymer. When there are contiguousblocks of the two or more repeating units in a polymer, the polymer is ablock co-polymer. As used herein the term polymer refers to a chemicalspecies containing a total of more than 10 repeating units (there may beone or more repeating units). The term oligomer is used herein to referto a chemical species having two to ten repeating units.

The conjugates of this invention are formed between a chemical specieswhich has at least one hydroxyl group or one thiol group and oligomersor polymers formed by ring-opening polymerization. The chemical speciesmust contain at least one functional group which under the conditions ofthe reaction functions as an initiator of the polymerization. Thehydroxyl group can most generally be a primary (1′), secondary (2′) ortertiary (3′) hydroxyl group attached to a carbon, or a hydroxyl groupattached to carbon of an aromatic ring which is generally describedherein as a “phenolic hydroxyl group.” Phenolic hydroxyl groups arethose directly attached to a carbon of an aryl ring. The terms hydroxyland hydroxy are used interchangeable herein. Hydroxyl groups do notinclude the OH moiety of —COOH groups (carboxylic acid groups) in whichthe hydrogen of the group is acidic. The hydrogens of phenolic hydroxylgroups are more acid than those of alcohols, but less acidic than thoseof carboxylic acid groups. The term hydroxyl as used herein also doesnot refer to —OH moieties which are bonded to N, P or S atoms. As isunderstood in the art a primary hydroxyl group is a hydroxyl groupbonded to a carbon atom that is also bonded to two hydrogens (e.g.,—CH₂—OH). A secondary hydroxyl group is a hydroxyl group bonded to acarbon atom that is bonded to one hydrogen atom (e.g. —CH(M)-OH, where Mis an atom or group other than H, in many cases M is a carbon containinggroup. A tertiary hydroxyl group is a hydroxyl group bonded to a carbonatom that is not bonded to a hydrogen, typically the carbon bonded tothe hydroxyl group is bonded to three other carbon atoms. The thiolgroup can most generally be a primary (1′), secondary (2′) or tertiary(3′) thiol group attached to a carbon, where the terms primary,secondary and tertiary are used as defined for the hydroxyl groups.

The particle formulation of this invention can be used to treat variousdiseases, disorders or conditions. Treatment methods of this inventioncomprise the step of administering a therapeutically effective amount ofthe drug to an individual in need of treatment in the form ofnanoparticles prepared by the methods of this invention containing thedrug. The term “therapeutically effective amount,” as used herein,refers to the amount a given drug that, when administered to theindividual in the particulate form, is effective to at least partiallytreat the disorder, disease or condition from which the individual issuffering, or to at least partially ameliorate a symptom of suchdisorder, disease or condition. As is understood in the art, thetherapeutically effective amount of a given compound will depend atleast in part upon, the mode of administration, any carrier or vehicle(e.g., solution, emulsion, etc.) employed, the specific disorder orcondition, and the specific individual to whom the compound is to beadministered (age, weight, condition, sex, etc.). The dosagerequirements needed to achieve the “therapeutically effective amount”vary with the particular compositions employed, the route ofadministration, the severity of the symptoms presented and theparticular subject being treated. Based on the results obtained instandard pharmacological test procedures, projected daily dosages ofactive compound can be determined as is understood in the art.

Particulate formulations herein can, for example, be in the form of drypowders which can be rehydrated as appropriate. The particulateformulations can be in unit dosage forms, e.g. in capsules, suspensions,dry powders and the like. In such form, the formulation can besub-divided in unit dose containing appropriate quantities of the activeingredient; the unit dosage forms can be packaged compositions, forexample, packaged powders, vials, ampoules, pre-filled syringes orsachets containing liquids. The unit dosage form can be, for example, acapsule, or it can be the appropriate number of any such compositions inpackage form.

The dosage employed can vary within wide limits and as is understood inthe art will have to be adjusted to the individual requirements in eachparticular case. Any suitable form of administration can be employed inthe method herein. The particles of this invention can be administeredin oral dosage forms, intravenously, intraperitoneally, subcutaneously,or intramuscularly, all using dosage forms well known to those ofordinary skill in the pharmaceutical arts. Compounds of this inventioncan also be administered in intranasal form by topical use of suitableintranasal vehicles. For intranasal or intrabronchial inhalation orinsulation, the compounds of this invention may be formulated into anaqueous or partially aqueous solution, which can then be utilized in theform of an aerosol.

The present invention provides methods of treating disorders, diseasesconditions and symptoms in a mammal and particularly in a human, byadministering to an individual in need of treatment or prophylaxis, atherapeutically effective amount of a particulate formulation of thisinvention to the mammal in need thereof. The result of treatment can bepartially or completely alleviating, inhibiting, preventing,ameliorating and/or relieving the disorder, condition or one or moresymptoms thereof. Administration includes any form of administrationthat is known in the art to be effective for a given type of disease ordisorder, and is intended to encompass administration in any appropriatedosage form. An individual in need of treatment or prophylaxis includesthose who have been diagnosed to have a given disorder or condition andto those who are suspected, for example, as a consequence of the displayof certain symptoms, of having such disorders or conditions.

The term drug includes “pharmaceutically acceptable salts” of drugs aswell as prodrugs The term “prodrug,” as used herein, means a compoundthat is convertible in vivo by metabolic means (e.g. by hydrolysis) to adrug.

Particles of the invention can be surface modified by any known methodto improve their surface properties for in vivo delivery or otherapplications. Particle surfaces can be modified for example bypegylation as is known in the art. Particle surfaces can be modified bycoating with a polymer as is known in the art.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. A number of specific groups of variabledefinitions have been described herein. It is intended that allcombinations and subcombinations of the specific groups of variabledefinitions are individually included in this disclosure. When acompound is described herein such that a particular isomer, enantiomeror diastereomer of the compound is not specified, for example, in aformula or in a chemical name, that description is intended to includeeach isomers and enantiomer of the compound described individual or inany combination. Additionally, unless otherwise specified, all isotopicvariants of compounds disclosed herein are intended to be encompassed bythe disclosure. For example, it will be understood that any one or morehydrogens in a molecule disclosed can be replaced with deuterium ortritium. Isotopic variants of a molecule are generally useful asstandards in assays for the molecule and in chemical and biologicalresearch related to the molecule or its use. Isotopic variants,including those carrying radioisotopes, may also be useful in diagnosticassays and in therapeutics. Methods for making such isotopic variantsare known in the art. Specific names of compounds are intended to beexemplary, as it is known that one of ordinary skill in the art can namethe same compounds differently.

Many of the molecules disclosed herein contain one or more ionizablegroups [groups from which a proton can be removed (e.g., —COOH) or added(e.g., amines) or which can be quaternized (e.g., amines)]. All possibleionic forms of such molecules and salts thereof are intended to beincluded individually in the disclosure herein. With regard to salts ofthe compounds herein, one of ordinary skill in the art can select fromamong a wide variety of available counterions those that are appropriatefor preparation of salts of this invention for a given application. Inspecific applications, the selection of a given anion or cation forpreparation of a salt may result in increased or decreased solubility ofthat salt.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein. References cited herein are incorporated by referenceherein to provide additional cyclic monomers, additional catalysts,additional reaction conditions, additional drugs and other chemicalspecies having at least one hydroxyl group, additional surface treatmentor modification methods and reagents and additional applications of themethods, compositions and kits of this invention.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, e.g., cyclic monomers, drugs and other chemical specieshaving at least one hydroxyl group, biological materials, reagents,e.g., ring-opening polymerization catalysts, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

EXAMPLES Example 1 Polylactide-Paclitaxel Nanoconjugate Particles

Nanoparticle design of this invention is based on the use of drugs asinitiators in ring-opening polymerization reactions to form drug-polymer(and drug-oligomer) conjugates in which the drug is covalently bonded tothe polymer or oligomer. Because the drug is used as the initiator ofpolymerization, the efficiency of conjugation of the drug to the polymer(oligomer) will be very high, ideally 100%. Additionally, if all of thedrug molecules are efficiently incorporated into a living polymerization(e.g. where drug molecules function as initiators), the drug loadingpercentage can be precisely controlled by adjusting themonomer/initiator ratio.

To initially demonstrate this strategy, paclitaxel (Ptxl) was used inthe presence of an appropriate catalyst to initiate a livingpolymerization of lactide. Utilization of molecules containing hydroxylgroups as initiators for the ring-opening living polymerization oflactide is well established. Paclitaxel, the best selling chemotherapydrug in the U.S., contains three hydroxyl groups.

Metal-oxides (M-ORs) are well-known initiators for living ring-openingpolymerizations of cyclic esters, such as DL-lactide (LA) used in thisstudy. They can be prepared in situ by mixing a hydroxyl-containingcompound with an active metal complex, such as a metal-amido compound(B. M. Chamberlain, M. Cheng, D. R. Moore, T. M. Ovift, E. B. Lobkovsky,G. W. Coates, J. Am. Chem. Soc. 2001, 123, 3229.) The in situ formedM-ORs can initiate controlled, living polymerization of LA, resulting inquantitative incorporation of OR to the PLA terminals and 100% monomerconversions. It was found that Ptxl can be incorporated into polyestersthrough metal-Ptxl oxide mediated polymerization of LA. Drug loadingscan thus be precisely controlled by adjusting LA to Ptxl ratios. Theincorporation efficiency of Ptxl to the resulting PLA should be 100% asthe formation of metal-OR is usually instantaneous and quantitative.After polymerization, Ptxl molecules are covalently linked to theterminals of PLA through a hydrolysable ester linker and are subject tosustained release upon hydrolysis. The Ptxl-PLA conjugates are employedin nanoprecipitation to generate polymeric NPs, Nanoconjugates (NC)containing covalently linked Ptxl.

To ensure a rapid and complete polymerization of LA (lactide) at roomtemperature (BDI)MgN(TMS)₂ (Chamberlain, et al. 2001 supra) a veryactive catalyst for the polymerization of LA was employed. (See: FIG. 2for structure of the catalyst.) Ptxl was mixed with 1 eq. (BDI)MgN(TMS)₂and the polymerization of LA was completed within minutes at roomtemperature with nearly quantitative incorporation of Ptxl into theresulting PLA (Table 1). It is believed that an in situ formed(BDI)Mg-Ptxl complex (structure uncharacterized; possibly a monomericMg-Ptxl oxide) initiated polymerization.

The Ptxl incorporated into the polymer conjugate was released to itsoriginal form and other degradation species after the Ptxl-PLA wastreated with 0.1-1 M NaOH, which demonstrated that Ptxl was conjugatedto PLA through a hydrolysable ester bond.

Nanoprecipitation of the Ptxl-PLA conjugates resulted in sub-100 nm NPs(Table 1). To be differentiated from NEs, these NPs derived fromnanoprecipitation of Ptxl-PLA conjugates are called nanoconjugates(NCs). Specific conjugated polymers are named herein as Drug (or otherchemical species)-LA_(n), where the drug or other chemical species isindicated by a shortened form (e.g., Ptxl, Dtxl, Doxo, Pyr or Cy5), andPLA is denoted as LA_(n) where n is the M/I ratio. In some cases, NC's,i.e., nanoprecipitates formed from the conjugated polymer are named NCof Drug-LA_(n). The use of these terms is clear from the context oftheir use.

NCs with monomodal particle distributions and low polydispersities wereconsistently obtained through the nanoprecipitation of Ptxl-PLAconjugates. Because the multimodal distribution of NEs is due in part tothe aggregation of the non-encapsulated free drug (J. Cheng, B. A.Teply, I. Sherifi, J. Sung, G. Luther, F. X. Gu, E. Levy-Nissenbaum, A.F. Radovic-Moreno, R. Langer, O. C. Farokhzad, Biomaterials 2007, 28,869), the monomodal distribution observed with NCs is likely related tothe unimolecular structures of Ptxl-PLA conjugates from which they aremade.

Both the solvent and the concentration of polymer have dramatic effecton the sizes of NPs prepared by nanoprecipitation. Solvent that hashigher water-miscibility (e.g., DMF) tends to diffuse into water fasterthan a solvent with lower water-miscibility (e.g., THF or acetone)(Cheng et al, 2007, supra). When a hydrophobic polymer in a highlywater-miscible solvent is added to water, fast nucleation of polymeraggregation is anticipated. Thus, the increased numbers of particles dueto rapid nucleation lead to reduced particle sizes when theconcentration of polymer remains unchanged in solution. When the solventtype and the solvent/water ratio are fixed, the particle sizes usuallyshow a linear correlation with the polymer concentrations because thenumber of particles remain roughly unchanged at that condition.

TABLE 1 Formation of drug-PLA nanoconjugates (NC) with high loadings,high incorporation efficiencies, small particle sizes and low particledistributions Load Lactide^(b) Particle Size + Polydispersity + Entry NCM/I^(a) (wt %) Conver. IE^(c) SD(nm)^(d) SD^(d,e) 1 Pyr-LA₁₀₀ 1001.6 >99% >99% 101.0 ± 1.4  0.09 ± 0.01 2 Pyr-LA₅₀ 50 3.1 >99% >99% 107.7± 2.2  0.07 ± 0.01 3 Pyr-LA₂₅ 25 6.1 >99% >99% 102.2 ± 1.0  0.06 ± 0.014 Ptxl-LA₁₀₀ 100 5.6 >99% >99% 95.1 ± 2.7 0.04 ± 0.01 5 Ptxl-LA₅₀ 5010.6 >99% >99% 80.6 ± 0.2 0.05 ± 0.01 6 Ptxl-LA₂₅ 25 19.2 >99%  97% 55.6± 0.5 0.04 ± 0.01 7 Ptxl-LA₁₅ 15 28.3 >99%  95% 85.5 ± 1.4 0.09 ± 0.03 8Dtxl-LA₁₀₀ 100 5.3 >99% >99% 84.7 ± 0.5 0.05 ± 0.02 9 Dtxl-LA₂₅ 258.3 >99%  98% 64.5 ± 0.7 0.05 ± 0.02 10 Dtxl-LA₁₀ 10 35.9 >99%  95% 77.9± 1.5 0.06 ± 0.02 11 Doxo-LA₁₀₀ 100 3.6 >99% >99% 96.3 ± 0.7 0.082 ±0.011 12 Doxo-LA₅₀ 50 7.0 >99% >99% 101.6 ± 0.6  0.075 ± 0.011 13Doxo-LA₂₀ 25 13.1 >99% >98% 90.8 ± 0.9 0.088 ± 0.010 14 Doxo-LA₁₀ 1027.4 >97% >94% 125.2 ± 2.3  0.110 ± 0.014 ^(a)M/I = monomer/initiatorratio. For all samples, they are first dissolved in DMF and dropwiseadded into water under rapid stirring; ^(b)Determined by analyzingunreacted lactide using FTIR (1771 cm − 1); ^(c)IncorporationEfficiency. Based on RP-HPLC analysis of free molecules. Incorporationefficiency is used instead of encapsulation efficiency as drug moleculesare conjugated to, not encapsulated in, polylactide; ^(d)Determined bydynamic light scattering, SD = standard deviation; ^(e)Whenpolydispersity is measured by the dynamic light scattering machine, thevalue is a statistical index to indicate the dispersity of the particlesize.

Nanoprecipitation of Ptxl-PLA conjugates followed these trends. At fixedconcentration of Ptxl-PLA conjugate, the sizes of NCs prepared byprecipitating a DMF solution of Ptxl-PLA conjugate are typically 20-30nm smaller than those prepared with acetone or THF as solvent. Whennanoprecipitation was carried out using DMF as solvent at a DMF/waterratio of 1/20 (v/v), the size of Ptxl-LA₂₀₀ NCs showed a linearcorrelation with the concentration of Ptxl-LA₂₀₀ conjugate, and can beprecisely tuned from 60 nm to 100 nm by changing the concentration ofPtxl-LA₂₀₀. Similar linear correlations were observed whennanoconjugates were formed with other drug-polymer conjugates bynanoprecipitation.

Compared with the conventional nanoprecipitates in which drug loadingand encapsulation efficiency can be extremely low in smaller particles,the nanoconjugates prepared by the method herein should all containexactly the same density of drug (e.g., Ptxl) in polymer matrix becausethe polymer-conjugate drug loading remains unchanged duringnanoprecipitation.

Drug burst release causes undesired side-effect and reduced therapeuticefficacy in nanoencapsulates. Since the Ptxl release kinetics ofPtxl-PLA NCs is determined by both the hydrolysis of the Ptxl-PLA esterlinker and the drug diffusion, the release kinetics of Ptxl from NCsshould be more controllable with significantly reduced burst releaseeffect. Well-controlled Ptxl release was observed in NCs (FIG. 3). Ptxlreleased from Ptxl-LA₅₀ (10.6 wt %) and Ptxl-LA₂₅ (19.2 wt %) were 7.0%and 8.7% at Day 1, and 43% and 70.4% at Day 6, respectively. Incomparison, 89% of Ptxl was released within 24 hrs from Ptxl/PLA NE(FIG. 3). The release of Ptxl from Ptxl-LA₅₀ NC was slower than thatfrom Ptxl-LA₂₅ NC, presumably because of the higher MW of Ptxl-LA₅₀ andmore compact particle aggregation. In fact, lower-loading NCs displayingslower drug release were observed in all drug-PLA NCs that were studied.

The in vitro toxicities of NCs are determined by the amount of Ptxlreleased; they thus show strong correlation with drug loadings (FIG. 4).The IC₅₀s of Ptxl-LA₁₅, Ptxl-LA₂₅ and Ptxl-LA₅₀ NCs with similar sizes(˜100 nm), determined by MTT assays in PC-3 cells, are 111, 370 and 855nM, respectively. The Ptxl-LA₁₅ NC has nearly identical IC₅₀ as freePtxl (87 nM); while the IC₅₀ of the Ptxl-LA₅₀ NC is an order ofmagnitude higher. As a result, the toxicity of NCs can be tuned in awide range simply by controlling NC drug loading.

Surface modification of NPs with poly(ethylene glycol) (PEG) is a widelyused approach for prolonged systemic circulation of NPs and reduced NPaggregation in blood. (P. Caliceti, F. M. Veronese, Advanced DrugDelivery Reviews 2003, 55, 1261; R. Gref, Y. Minamitake, M. T.Peracchia, V. Trubetskoy, V. Torchilin, R. Langer, Science 1994, 263,1600.)

A non-covalent approach to pegylate the NC surface was initiallyemployed to reduce the efforts of removing unreacted reagents andby-products. For example, poly(glycolide-co-lactide)-b-methoxylated PEG(PLGA-mPEG), an amphiphilic copolymer that has a 13 kDa PLGA and a 5 kDaPEG segment was used to pegylate the NCs. It has been reported by Pierriet al. (Journal of Biomedical Materials Research Part A; 2005; 639-647)that the micellation of amphiphilic copolymer PLA-b-PEG can besignificantly eliminated when PEG is above 70% or below 50% of thehydrophobic block.

It is expected that the PLGA block forms strong interaction with NCsthrough hydrophobic interaction to create a stable PEG shell. Similarapproach has been used previously in NP surface pegylation. (X. H. Gao,Y. Y. Cui, R. M. Levenson, L. W. K. Chung, S. M. Nie, Nat. Biotechnol.2004, 22, 969). Sequential addition of 0.4 to 2 equivalent (in mass) ofPLGA-mPEG to Ptxl-LA₂₀₀ resulted in a linear increase in particle sizefrom 54.5 nm to 100.3 nm (FIG. 5).

The PLGA-mPEG modified Ptxl-LA₂₀₀ NCs showed significantly enhancedstability in PBS compared to the untreated NCs or NCs treated only withmPEG (FIG. 6), indicating the importance of the hydrophobic PLGA segmentto the non-covalent interaction between PLGA-mPEG and NCs. NCs aresubject to instantaneous dilution after intravenous administration,which may result in dissociation of PLGA-mPEG from NCs. However,sequential dilution of the PLGA-mPEG treated Ptxl-LA₂₀₀ NCs from 1 mg/mLto 0.01 mg/mL did not show any increase in particle size in PBS. Thisstudy indicates that the PEG shells formed as described above shouldremain tightly bound to the NCs in systemic circulation.

Surface coating of NCs with PEG can lead to formation of longcirculating nanoparticles. The linear increase of nanoparticle sizeobserved when PLA-PEG was added to the Ptxl-PLA nanoparticles indicatesthe formation of a layered structure on the surface of the nanoparticlebecause of the hydrophobic interaction of PLA-PEG and PLA-paclitaxel.PEG corona were formed readily. Nanoparticles generated using thismethod are stable in salt solution. The simple strategy of makingsalt-stable nanoparticles will make the nanoparticles readilytransferable to systemic study.

PEG can also be covalently conjugated to the NCs as is known in the art.(O. C. Farokhzad, J. J. Cheng, B. A. Teply, I. Sherifi, S. Jon, P. W.Kantoff, J. P. Richie, R. Langer, Pro. Nat'l Acad. Sci. USA 2006, 103,6315).

Ptxl has three hydroxyl groups at its C-2′, C-1 and C-7 positions,respectively. Each of these three hydroxyl groups can potentiallyinitiate LA polymerizations, resulting in Ptxl-PLA conjugates with 1 to3 PLA chains attached to Ptxl. To reduce the heterogeneity of Ptxl-PLA,it is preferred that Ptxl-PLA conjugate containing a single PLA chain.It has been found that polymerization initiation can be controlled at aspecific hydroxyl group of the drug (e.g., Ptxl) to make the PLAconjugate containing a single PLA chain.

The three hydroxyl groups of Ptxl differ in steric hindrance in theorder of 2′-OH<7-OH<1-OH. The tertiary 1-OH is least accessible andtypically is inactive. (D. Mastropaolo, A. Camerman, Y. G. Luo, G. D.Brayer, N. Camerman, Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6920.) The7-OH, however, could potentially compete with the 2′-OH, the mostaccessible and active hydroxyl group of Ptxl, for complexing with metalcatalyst. In view of the difference in steric hindrance at the differentOH groups, it was postulated that employing a catalyst with bulkygroups, e.g., a metal catalyst with a bulky chelating ligand mightdifferentiate between 2′- and the 7-OH, and thus preferentially or evenspecifically form Ptxl-metal complex through the 2′-OH for LApolymerization.

Attempts using NMR to determine to which hydroxyl group(s) the PLAchains were attached were unsuccessful because of the complexity of thematerial. To determine if PLA was attached to the 2′-OH or the 7-OH, thePtxl of Ptxl-PLA was reacted with tetrabutylammonium borohydride(Bu₄NBH₄). This reagent is reported (N. F. Magri, D. G. I. Kingston, C.Jitrangsri, T. Piccariello, J. Org. Chem. 1986, 51, 3239) toquantitatively reduce the 13-ester bond of Ptxl into Baccatin III (BAC)and (1S,2R)—N-1-(1-phenyl-2,3-dihydroxypropyl)benzamide (PDB). BACcontains the 7-OH of PTxl, while PDB contains the 2′-OH of Pxtl.

Ptxl-LA₅ was prepared using different metal catalysts: Mg(N(TMS)₂)₂, acatalyst without a chelating ligand, and (BDI)MgN(TMS)₂ with a chelatingligand and reacted with Bu₄NBH₄. With Mg(N(TMS)₂)₂, both PDB-PLA andBAC-PLA resulted from the reaction indicating that with this catalystpolymerization initiated at both the 2′-OH and the 7-OH of Ptxl. When(BDI)MgN(TMS)₂ was used, the amount of BAC-PLA derived was significantlyreduced indicating polymerization of LA was with this catalystpreferentially initiated at the 2′-OH of Ptxl.

Although (BDI)MgN(TMS)₂ gave significantly improved site-specificcontrol in the metal/Ptxl initiated polymerization, the resultingPtxl-PLAs typically have fairly broad MWD (e.g., Ptxl-LA₂₀₀M_(w)/M_(n)=1.47). This observation was attributed to fast propagationrelative to initiation for polymerization initiated by Mg catalysts.(Chamberlain et al., 2001 supra). The catalyst (BDI)ZnN(TMS)₂, has achelating ligand identical to that of (BDI)MgN(TMS)₂, but is lessreactive compared to its Mg analogue even though it gives significantlyimproved polymerization of LA. When (BDI)ZnN(TMS)₂ was used inPtxl-initiated LA polymerization with M/I=200. The resulting Ptxl-LA₂₀₀had an extremely narrow PDI (M_(w)/M_(n)=1.02) and the MW M_(n)(obtained)=28,100 kDa; compared to M_(n) (expected)=29, 700 kDa). HPLCanalysis of Ptxl-LA₅ that was prepared with (BDI)ZnN(TMS)₂ and thentreated with Bu₄NBH₄ as described above demonstrated that initiation andpolymerization was exclusively at the 2′-OH of Ptxl6

Drug-initiated polymerization method can be applied to the preparationof NCs of other hydroxyl-containing therapeutic agents, as well as todrugs containing one or more thiol groups. For instance, docetaxel(Dtxl)-LA₁₀ and camptothecin (CPT)-LA₁₀ NCs with very high drug loading(35.9 wt % and 19.5 wt %, respectively), more than 95% loadingefficiencies and sub-100 nm sizes are readily prepared using thismetal/drug complex initiated LA polymerization followed bynanoprecipitation (Table 1). CPT differs from both Ptxl and Dtxl as ithas no intrinsic ester bond. It thus was quantitatively recovered fromCPT-PLA NC after being treated with NaOH. The CPT separated from thehydrolysis mixture of CPT-PLA in PBS and collected on a preparative HPLCshowed a 1H NMR spectrum identical to that of the authentic CPT. Thisstudy further demonstrated that the chemical structures of theincorporated drugs remain unchanged under the mild polymerization andnanoprecipitation processes. The incorporated drugs in NCs can bereleased to their original forms.

Like Ptxl-LA NCs, Dtxl-LA NCs, Doxo-LA and CPT-PLA NCs showed no burstrelease effects in PBS. The toxicity-drug loading correlations of bothDtxl-PLA and CPT-PLA NCs are similar to that of Ptxl-PLA NCs (FIGS.7A-D).

The method of this invention allows preparation of polymer-drugconjugated NPs (NCs) that have very high drug loadings (up to 35%),nearly quantitative loading efficiencies, controlled release profileswithout burst release effects, and narrow particle distributions. Safe,nutrient metals are involved in this formation; and organic chelatingligand can be readily removed via solvent-extraction. Additionally ittakes only a few hours to prepare gram-scale, high-loading, salt-stableNCs. Drug release profiles can potentially be further controlled throughthe use of different cyclic ester monomers, as discussed above, forpreparing NCs.

Preparation of Ptxl-PLA NC: (BDI)MgN(SiMe₃)₂ (6.2 mg, 0.01 mmol) andPtxl (8.5 mg, 0.01 mmol) were mixed in 0.5 mL anhydrous THF. DL-Lactide(144 mg, 1 mmol) in 2 mL anhydrous THF was added dropwise. After LA wascompletely consumed (monitored by FT-IR or 1H NMR), the polymerizationsolution was precipitated in ethyl ether (25 mL) to give Ptxl-LA₁₀₀conjugate. Polymerization using (BDI)ZnN(SiMe₃)₂ or Mg(N(SiMe₃)₂)₂ weresimilarly performed. An acetone or DMF solution of Ptxl-LA₁₀₀ (100 μL,10 mg/mL) was precipitated dropwise to vigorously stirred nanopure water(2 mL). PLGA-mPEG5k (MW=18,300 g/mol, 5 mg/mL in DMF, 100 μL) or mPEG5k(5 mg/mL in DMF, 100 μL) was dropwise added to NCs.

Characterization and Evaluation of Ptxl-PLA NC: NC sizes werecharacterized by a ZetaPALS dynamic light scattering detector(Brookhaven Instruments, Holtsville, N.Y., USA) or by SEM. The resultingNCs were purified by ultrafiltration; their in vitro toxicities werethen evaluated using MTT assay in PC-3 cells (24 hr incubation at 37 C).To determine the release kinetics of Ptxl-PLA, a PBS solution of NCs wasequally divided into several portions and then incubated at 37 C. Atscheduled time, the release study was terminated. DMF solution was addedto dissolve all precipitation. All the samples were then dried,re-dissolved in DCM and reacted with Bu₄NBH₄ for 1.5 hours. A drop ofacetic acid was added into the solution. The solution was stirred for 20min before the solvent was evaporated. The residual solid wasreconstituted in acetonitrile for RP HPLC analysis (Curosil, 250×4.6 mm,5μ; Phenomenex, Torrence, Calif., USA).

We have demonstrated that conjugate formation using the strategyillustrated in FIG. 2 for several different small molecules and for apeptide. The structures of these molecules are shown in FIG. 8.Representative nanoconjugate data are shown in Table 1. Exemplarydetailed PLA-drug polymerization conditions are provided below.Nanoparticle formation, characterization, and release evaluation werecarried out similarly to those methods used for PLA-paclitaxelnanoparticles.

Example 2 Polylactide-Doxorubicin Polymer (Doxo-PLA)

The catalyst (BDI)MgN(TMS)₂ and D,L-lactide were treated as in Ptxl-PLApolymerization. The polymerization was conducted in a glove box. All thereaction vessels were covered with aluminum foil and the box light wasturned off. First, doxorubicin was dissolved in DMF and stirred for 10min, until it completely dissolved. (BDI)MgN(SiMe₃)₂ was then added toand dissolved in THF. The doxorubicin and (BDI)MgN(SiMe₃)₂ solutionswere mixed for 15-20 min, and the solution changed color from orange redto purple. On HPLC analysis, the peak associated with doxorubicinshifted, indicating that a complex of (BDI)MgN(SiMe₃)₂ and doxorubicinwas formed. The UV detector was at 450 nm. D,L-lactide was dissolved inTHF and added dropwise into the mixture of doxorubicin and(BDI)MgN(SiMe₃)₂ with rapid stirring. The reaction process was monitoredby HPLC until all of the doxorubicin was gone. The UV spectrum ofDoxo-LA exhibited an absorption at 325-400 nm, different from theabsorption of doxorubicin at 400-500 nm. Nanoparticles were formed bynanoprecipitation similarly to Ptxl-PLA nanoparticles.

Example 3 Dtxl-PLA using 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) orBDI—Mg—N(TMS)₂

The TBD or BDI—Mg—N(TMS)₂ catalyst was mixed with docetaxel and thelactide was added to the mixture of catalyst and initiator. For example,docetaxel and TBD were dissolved in THF solution and stirred for 5-10min. (In HPLC, the peak of docetaxel shifted, indicating the TBD formedcomplex with docetaxel). D,L-Lactide was dissolved in THF solution andadded dropwise into the mixture of docetaxel and TBD. The reaction wassimilar to that of paclitaxel-PLA, monitored by FTIR and HPLC.Polymerization initiated by docetaxel-Mg(II) complex was carried out inthe same way as described above for paclitaxel. Nanoparticles wereformed similarly to Ptxl-LA nanoparticles.

Example 4 PLA-Pyrenemethoxy Polymerization

Pyrenemethanol (TCI America) was purified by dissolving in THF with CaH₂and stirring overnight, filtered and vacuum dried and stored in glovebox freezer. The pyrenemethanol was mixed with the LA in BDI—Mg—N(TMS)₂.The ratio of monomer to initiator (Pyr) was selected. After 24 hours thereaction was stopped and purified by washing with methanol and ether forthree times. NMR confirmed the PLA copolymer formation conjugated withpyrenemethanol. Nanoparticles were formed similarly as Ptxl-PLAnanoparticles.

Examples 5 PLA-LHRH Polymerization

Goserelin was obtained from Bachem and stored in a freezer.(BDI)MgN(SiMe₃)₂ and D,L-Lactide were treated as described above forPtxl-PLA polymerization. The polymerization was conducted in a glovebox. Goserelin was dissolved in DMF solution with stirring for 10 min.(BDI)MgN(SiMe₃)₂ was dissolved in THF solution. The goserelin and(BDI)MgN(SiMe₃)₂ solutions were mixed for 30 min with stirring.D,L-Lactide was dissolved in THF and added into the mixture. The ratioof lactide to goserelin was selected. During polymerization, thesolution may become cloudy indicating precipitation of some species. Ifthis occurs, more DMSO can be added to keep the components and productsin solution. The conversion of goserelin was detected by HPLC. Theconversion observed was however less than 95%. Nanoparticles were formedfrom the groserelin-conjugates as described for Ptxl-PLA nanoparticles.

Example 6 Zn, Ca and Fe Catalysts, and Organocatalysts

Mg(II) complexes gave fast polymerization. However, in some instancesMg(II) may give significantly faster propagation than initiation whichis undesirable for carrying out a living polymerization. Coates hasdemonstrated that certain Zn catalysts facilitate fast initiation andrelatively slow chain propagation and Zn-mediated lactide polymerizationcan result in polymers with narrow polydispersity.¹⁹ Therefore,(BDI)Zn-(docetaxel) and (BDI)Zn-(doxorubicin) are useful as initiatorsin the methods herein. Other catalysts useful for these methods includethose of Ca and Fe.¹⁶ Mg, Zn, Ca, and Fe are elements found in humanbody, therefore they have should better safety profile than other activecatalysts such as Al and Sn. Exemplary catalyst include, among others,

10

12

13 Ar = 2,6-(i-Pr)₂C₆H₃

22a M = Zn, X = OSiPh₃ 22b M = Mg, X = OPh

23

21 21′ M = Mg, Ca, Zn X = OEt, OPh, O[2,6-(i-Pr)₂C₆H₃], OSiMe₃ N(SiMe₃)₂R = t-Bu, i-Pr

complex R¹ R² 48a Me H 48b Me Me 48c Me t-Bu 48d Me Cl 48e CH₂Ph H 48fCH₂Ph Me 48g CH₂Ph t-Bu 48h CH₂Ph Cl L₁ZnN(SiMe₃)₂ (29a) L₁ZnN(i-Pr)₂(29b) (L₁ZnOi-Pr)₂ (29c) L₁Zn(OSiPh₂)(THF) (29d) L₁ZnOt-Bu (29e)L₁ZnOCHMeCO₂Me (29f) (L₁ZnOAc)₂ (29g) L₁ZnEt (29h) L₁SnOi-Pr (30)L₁Mg[N(i-Pr)₂](THF) (31a) (L₁MgOi-Pr)₂ (31b) L₁Mg(Ot-Bu)(THF) (31c)L₁Ca[N(SiMe₂)₂](THF) (32) L₂FeOt-Bu (33), where

Ar = 2,6-(i-Pr)₂C₆H₃ or Ar = 2,6-(Ethyl)₂C₆H₃

Example 7 Release Studies Pyrenemethanol-PLA Release Study

Free pyrenemethanol was used to calibrate the concentration-HPLC peakarea (or intensity) curve. The nanoparticle was formed in acetonitrile(10 mg/ml)-water (1/10) system and washed by DI water three times toremove non-covalently bound small molecules. At day 0, the PBS 1X-NPsolutions were prepared in several vials and incubate at 37 C. Two vialswere used in an experiment. The content of one vial was centrifuged at4000 rpm for 30 min to spin down all the NPs and the concentration ofpyrenemethanol concentration in the supernatant was measured. 1N NaOHsolution was added to the other vial at 37 C for 30-90 min to degradeall the NPs and the total concentration of pyrenemethanol (100%) wasmeasured. (Before injection into the HPLC, the pH value of the solutionwas adjusted to 7 using acetic acid). At a selected time point, a vialis taken from the 37 C incubator, NPs are spun down to get thesupernatant and prepare to 1/1 PBS-acetonitrile solution and injectedinto HPLC. The integrated peak and intensity was documented and comparedwith measurement of total 100% pyrenemethanol concentration in thenanoparticles to determine the release profile. The HPLC detectormonitored for absorption at 227 nm and 265 nm. The mobile phase used is50/50 acetonitrile/DI water with 0.05% TFA.

Docetaxel-PLA Release Study

Free Docetaxel was used to calibrate a standard curve in HPLC analysis.The PLA-docetaxel nanoparticles were well dispersed in 1×PBS solutionand extracted with 1-octanol. The 1-octanol extract was directlyinjected into the HPLC. The analysis condition used were the same asused for PLA-pyrenemethanol, and the detector monitored at 227 nm and265 nm. The percentage of release is determined by the comparison of theamount of docetaxel in 100% incorporation polymer at the same HPLCinjection concentration.

Example 8 Dendritic Nanoconjugates in 2-20 nm Range

Because drug release kinetics are directly correlated to nanoparticlesurface area, further miniaturization of nanoparticles will result ineven faster drug lease. In addition, small nanoparticles can havedramatically different properties for drug delivery and otherapplications compared to larger particles. For example, smallnanoparticles should have different cell uptake characteristics. Forrelatively large particle size (˜100 nm), particles can be endocytosed.But when the NP is in ˜10 nm range, direct particle penetration or entrythrough solution pinocytosis (cell drinking) is possible. Micellation ofblock copolymers typically results in particles larger than the 10 nmrange.

To prepare smaller size nanoparticles, dendritic polymer or oligomerconjugates can be used. Such conjugates are formed by polymerization AB2type cyclic monomers, such as hydroxyl lactides:

where z is 1-6 and Y₁ is as defined above. In specific embodiments Y₁ isH.

For example, the AB₂ monomer of Formula H where z is 1 and Y1 is H canbe used. This monomer is synthesized as illustrated in Scheme 1 below.Polymerization of the hydroxyl lactide molecule in the presence of adrug having at least one hydroxyl or thiol group results in formation ofa conjugate having a dendritic or hyperbranched structure as illustratedin FIG. 9.

In another specific embodiment, in which the drug or other chemicalspecies has one hydroxyl group, a single attached polymer can form ahyperbranched structure around the drug molecule. In a specificembodiment, in which the drug or other chemical species has two or morehydroxyl groups, multiple attached polymers can form a hyperbranchedstructure around the drug molecule. This embodiment is exemplified inFIG. 9. It will be appreciated, that not all hydroxyl groups in the drugor other species, will have the same reactivity for initiation ofpolymerization and attachment of the polymer or oligomer. Thus, for agiven drug or other chemical species, the polymer or oligomer may beformed selectively at fewer than all hydroxyl groups. Also a givenpopulation of polymer/oligomer conjugates may be heterogeneous withrespect to the number of polymers/oligomers attached to each drug orother chemical species, i.e., it may be that not all of the conjugateswill have the same number of attached polymer/oligomers. The formationof sufficient hyperbranched structure results in the formation of aunimolecular dendritic nanoparticle. This method employing a cyclic AB2type monomer can result in the formation of small particle sizenanoparticles (20 nm or less) containing a selected drug. The resultingnanoparticles are water soluble as the hyperbranched polyester structurehas many peripheral hydroxyl groups.

In the synthesis of Scheme 1, H-Ser(Z)-OH (30.0 mg, 154 mmol) wasdissolved in 1M sulfuric acid 400 ml and acetonitrile 400 ml. NaNO₂(21.7 g 313 mmol) was dissolved in 150 ml water and added dropwise intothe reaction flask over 30 min. The reaction was stirred for 18-24 hoursunder nitrogen protection. The solution was extracted withdichloromethane and ethyl acetate 500 ml (3 times). The combined organiclayers were dried with magnesium sulfate, filtered, and concentrated invacuo. The resulting product (25.4 g, 129 mmol) was dissolved indichlormethane (300 ml) with 17.9 ml triethylamine and added dropwise(over 30 minutes) into 2-bromopropionyl bromide (13.5 ml 129 mmol) andDMAP (1.58 g, 129 mmol) in 150 ml dichloromethane solution in an icecold bath. The reaction was thereafter stirred for 18-24 hours undernitrogen. The mixture was precipitated using ether and the remainingsolution was filtered. The combined organic solution was evaporated togive a light yellow oil (product of second step)

Sodium iodine (18.59 g, 1.24 mol, 10 equivalents) was added into 500 mlacetone solution containing the product of the second step (4.1 μg, 12.4mmol) and the mixture was refluxed overnight. Temperature was controlledto 55-60 C under nitrogen. The reaction was stopped and cooled down thenext day. Solvent (acetone) was filtered using celite and the acetonewas evaporated. The resulting brown oil was redissolved in acetone andextracted with Na₂S₂O₃ 2M (300 ml)(3 times) to give a yellow solutionwhich was dried with MgSO₄. Finally the solvent was evaporated to give acrude product (of step three) which was used without furtherpurification.

A solution of this product (4.53 g 12.1 mmol) in 100 ml DCM was addeddropwise to refluxing DIEA (4.6 ml, 27.7 mmol) in 1000 ml acetone over 8hours. The temperature was controlled at 70 C. HPLC confirmed that thereaction was complete. The diastereomers were separated on a silica gelcolumn by eluting with chloroform and methyl tert butyl ether (1/1). Thesolvent was evaporated and the solid was redissolved in methyl tertbutyl ether, and then excess hexane was added to the solution toprecipitate out a white solid.

The white solid (1.5 mmol, 375.0 mg) was dissolved in methanol (1.5 m)and the Cbz group was deprotected by H₂ with 10% Pd/C catalyst (40 mg).After two days the solution was evaporated to give a white solid. NMRand EI-MS confirmed the structure.

The polymerization of the hydroxyl substituted cyclic monomer (1.0 mmol,160 mg) is conducted in a glove box. The monomer is dissolved in 500 ulDMF solution. (BDI)MgN(SiMe₃)₂ (0.03 mmol, 18.0 mg) is dissolved in 1 mlTHF and mixed with docetaxel (0.01 mmol, 8.1 mg) in THF solution for10-30 min. The monomer solution is very slowly added into the mixtureover 10 min. The reaction is monitored by HPLC at 227 nm and 265 nm.After finishing the reaction, the polymer is purified by reverse-phasesemi-preparative HPLC using a SEC column. The polymer forms micelles inwater solution and the size of the particles formed is in the range from5-30 nm, as characterized by AFM and dynamic light scatteringmeasurement.

Example 9 Design of Nanoconjugates in 20-60 nm Range

It is a challenge to make nanoparticles smaller than 60 nm, particularlyby precipitating hydrophobic PLA or Dtxl-PLA. Micellation can, however,be used to make nanoparticles with precisely controlled sizes in a rangeof 20-60 nm. PEG is conjugated to the terminal —OH group of Dtxl-LA. Theconjugation of PEG can be performed after the polymerizationconjugation. Alternatively, a single reaction step can be employed.After each polymerization, the terminal hydroxyl group is reactive tocapping groups like isocyanate. PEG-isocyanate, for example, is used asa capping agent to cap the terminal hydroxyl groups of drug-PLAconjugate. PEG-isocyanate is readily prepared by reacting PEG-NH₂ withtriphosgene. Reaction of PEG-isocyanate with Dtxl-LA-OH is fast, andresults in Dtxl-LA-urethane linker-PEG in quantitative yield. Afterpurifying the conjugated polymer through an SEC column, the purifiedconjugated polymer is used to formulate micelles. When PEG with higheraverage MW is used, micelles with small particle size (20 nm-60 nm) canbe readily generated. The use of PEG-poly(aspartic acid)-drug conjugatesto form copolymer micelles has been extensively studied by Kataoka,²⁰Kwon^(21, 22) and the inventors.^(23, 24)

In a specific example, mPEG5k-NH₂ 500 mg (0.1 mmol) was dissolved indichloromethane (10 ml) with stirring. Triphosgene 269.8 mg (1 mmol) wasadded to the solution and the reaction mixture was refluxed at 50-60 Cfor at least 2 hours. The reaction was monitored by FTIR, where theisocyanate peak appears at 2250 cm⁻¹. The yield is calculated by thetitration of the mPEG5k-NCO with the quantified pyrenemethanol todetermine the concentration of the mPEG5k-NCO in the solution. After thereaction is complete, the solvent is evaporated and the polymer iswashed with hexane and cold ether (3 times). Unreacted mPEG5k-NH₂ isseparated from the product by a size exclusion column using THF oracetonitrile/hexane as eluent. The product is dried in vacuo and storedunder a nitrogen atmosphere in the freezer.

Example 10 Double Emulsion Technique Design of Nanoparticles (100-600nm), Exemplified with Dtxl-LA Conjugates

Drug encapsulated microparticles are prepared using awater-in-oil-in-water (W/O/W) solvent evaporation procedure. In brief,50 microliter of water was emulsified in a 1 mL solution of the polymerconjugate (Dtxl-LA) (50 mg) in dichloromethane using a probe sonicator(Sonic & Materials Inc, Danbury, Conn., USA) at 10 W for 15-30 s. Theemulsion was then poured into 50 mL of aqueous PVA (1%) or sodiumcholate solution (1% w/v) and the mixture was homogenized for 1 min(8000 rpm). The resulting emulsion was poured into 150 mL of aqueous PVAor sodium cholate solution (0.3% w/v) with gentle stirring, after whichorganic solvent was evaporated by stirring at RT for 2 h or rapidlyremoved using a rotary evaporator. Finally, the nanoparticles wereisolated by centrifugation at 6000 rpm for 30 min, washed with distilledwater, and preserved at −15 C in an emulsion form in distilled water (6mL). Alternatively, the nanoparticles can be lyophilized to obtain apowder.

Example 11 Double Emulsion Method for Preparation of Microparticles (600nm-100 μm)

Drug encapsulated microparticles are prepared using thewater-in-oil-in-water (W/O/W) solvent evaporation procedure (doubleemulsion method) employed elsewhere. In brief, 50 microL of water isemulsified in a 1 mL solution of the polymer conjugate (Doxo-LA) (50 mg)in dichloromethane using a probe sonicator (Sonic & Materials Inc,Danbury, Conn., USA) at 10 W for 15-30 s. The emulsion is then pouredinto 50 mL of aqueous PVA (1%) or sodium cholate solution (1% w/v) andthe mixture is homogenized for 1 min at a speed of 500-8000 rpm. Theresulting emulsion is poured into 150 mL of aqueous PVA or sodiumcholate solution (0.3% w/v) with gentle stirring, after which organicsolvent is evaporated by stirring at room temperature for 2 h or israpidly removed using a rotary evaporator. Finally, the nanoparticlesare isolated by centrifugation at 6000 rpm for 30 min, washed withdistilled water, and preserved at −15 C in emulsion form in distilledwater (6 mL) or are lyophilized to obtain a powder.

Example 12 Surface Functionalization Using Herceptin

Trastuzumab (Herceptin) is dissolved at 1 mg/ml in phosphate buffer(pH=8.0). 2-iminothiolate (5.7 mg) is dissolved in 5 ml phosphate bufferat pH 8.0. The 2-iminothiolate solution (8.04 microL) is mixed with 1 mlTrastuzumab solution for 6 hours at 20 C. The resulting thiolatedantibody can be purified using a Dextran Desalting SEC column withphosphate buffer as eluent and detecting at 280 nm. The antibodysolution is further concentrated using a Microcon 30000microconcentrator. The thiol group concentration in the antibodysolution can be determined using Ellman reagent. The antibody solution250 ul is mixed at room temperature with 6.25 ul 4 mg/ml Ellman reagent(in phosphate buffer pH=8.0) for 15 min, and detected by the UVspectrometer at 412 nm. The number of thiol groups is calculatedrelative to a L-cysteine standard solution.

MAL-PEG5k-isocyanate (where, MAL is a malimide group) is conjugated toDoxo-PLA at room temperature for 8 hours and the polymer is purified onan SEC column. The polymer is dissolved in acetone at 10 mg/mlconcentration and added dropwise under vigorous stirring into water withthe volume ratio of acetone:water=1/20. The coupling reaction of thiolof the antibody and the malimide of the polymer is then conducted innanopure water solution at room temperature. The reaction is allowed tocontinue for at least 12 hours. The SEC column is used to detectunbounded thiolated antibody in the water phase. After conjugation, theefficiency is determined by compared the peak area of thiolated antibodyfrom the SEC column before reaction at the same concentration.

There is an alternative method for incorporation of thiolated antibody.The PLGA-PEG-MAL (poly(lactic-glycolic acid)-poly(ethylene glycol)copolymer with a terminal malimide group) or PLA-PEG-MAL (poly(lacticacid)-poly(ethylene glycol) copolymer with a terminal malimide group)can either be purchased or made by known methods. First the Doxo-LA isdissolved in acetone and the material is nanoprecipitated to formnanoparticles. Then an acetone solution of PLGA-PEG-MAL or PLA-PEG-MALis added dropwise to the nanoparticle. Use of this method keeps theparticle distribution unchanged. The further conjugation between themalamide and thiol group of the antibody is performed as describedabove.

After conjugation, the nanoparticle is surface-modified with theherceptin. The function of the antibody can be tested. The nanoparticlesare incubated with MCF-7 and SK-BR-3 cells for 3 days after whichwestern blotting analysis is performed. The SK-BR-3 cell that expressHER 2 and MCF 7 cells are used as a negative control.

Example 13 Preparation of Cyclopamine (Ca) Ncs

The preparation of CA-LA₁₀₀ nanoconjugates involved two steps. The firststep was to conjugate CA with PLA polymer. Briefly, in the glove box,cyclopamine (4.11 mg, 0.01 mmol) was dissolved in 1.0 mL THF solution.(BDI)MgN(SiMe₃)₂ (6.0 mg, 0.01 mmol) was mixed with CA for 5-15 min.DL-Lactide (144 mg, 1.0 mmol) in 1 mL THF solution was added dropwise tothe mixture of CA and (BDI)MgN(SiMe₃)₂ with vigorous stirring. FTIR wasused to calculate the conversion of Lactide. The reaction finishedovernight and CA-LA₁₀₀ conjugated polymer was obtained. The second stepwas to use the polymer solution to directly prepare NPs by thenanoprecipitation method. CA-LA₁₀₀ polymer in DMF solution was addeddropwise into 20× nanopure water, a non-solvent. The resulting NPssuspension can be purified by ultrafiltration (15 min, 3000 g, AmiconUltra, Ultracel membrane with 10,000 NMWL, Millipore, Billerica, Mass.).

Example 14 Preparation of Cyclosporin (Cp) Ncs

The preparation of CP-LA₁₀₀ nanoconjugates involved two steps. The firststep was to conjugate CP with PLA polymer. Briefly, in the glove box,cyclosporine 12.02 mg (0.01 mmol) was dissolved in 1.0 mL THF solution.(BDI)MgN(SiMe₃)₂ (6.0 mg, 0.01 mmol) was mixed with CA for 5-15 min.DL-Lactide (144 mg, 1.0 mmol) in 1 mL THF solution was added dropwise tothe mixture of CA and (BDI)MgN(SiMe₃)₂ with vigorous stirring. FTIR wasused to calculate the conversion of Lactide. Finally the reactionfinished overnight and CA-100 polymer was obtained. The second step wasto use the polymer solution to directly prepare NPs by nanoprecipitationmethod. In general, CA-100 polymer in DMF solution was dropwise addedinto 20×nanopure water, a non-solvent. The resulting NPs suspension canbe purified by ultrafiltration (15 min, 3000 g, Amicon Ultra, Ultracelmembrane with 10,000 NMWL, Millipore, Billerica, Mass.).

Example 15 Preparation of Core-Shell Nanoconjugates (50-200 nm Range)Including Those Containing Two or More Drugs

Doxo-LA₂₅ polymer (5 mg/mL in DMF, 100 μL) was added dropwise to 2 mLnanopure water to give Doxo-LA₂₅ NCs. PLGA-mPEG5k (MW=18,300 g/mol, 5mg/mL in DMF, 100 μL) or mPEG5k (5 mg/mL in DMF, 100 μL) was then addeddropwise to Doxo-25 NP. The NC that resulted are core-shell NCs wherethe core is Doxo-LA25 and the shell is PLGA-mPEG5k or mPEG5k.

Ptxl-LA₂₀₀ conjugate (2 mg/mL in DMF, 100 μL) was added dropwise to 2 mLnanopure water to give Ptxl-LA₂₀₀ NCs. PLA-PEG3k (MW=17,500 g/mol, 2mg/mL in DMF) was then sequentially added into Ptxl-LA200 NCs solutionunder vigorous stirring. The resulting NCs are core-shell NCs.

Analogous methods can be employed with drug-polymer conjugate preparedby the methods herein to form analogous nanoconjugates with core-shellstructure.

Dtxl-LA₁₀₀(core)/Doxo-LA₁₀₀ (Shell) NCs: Dtxl-LA₁₀₀ in DMF (10 mg/mL,100 μL) was added dropwise into 2 mL nanopure water to formnanoparticles (NCs) and thereafter Doxo-LA₁₀₀ in DMF (10 mg/mL, 100 μL)was further added into the nanoparticle solution with vigorous stirring(3000 rpm) at 50 μL/min rate. The resulting NCs are dual drug core-shellNCs illustrated in FIG. 10A. In this figure the shift in particle sizefrom about 80 to about 100 nm is illustrated.

Ptxl-LA₁₀₀/CPT-LA₁₀₀: Ptxl-100 in DMF (10 mg/mL, 100 μL) was addeddropwise into 2 mL nanopure water and the CPT-100 in DMF (10 mg/mL, 100μL) was further added into the nanoparticle solution with vigorousstirring (3000 rpm) at 50 μL/min rate.

These methods can be employed to prepare NCs containing multiple drugsemploying any drug-polymer conjugate made by the methods herein. Themethods can also be employed to prepare NCs where the core and shell aremade from different polymer conjugates with the same drug e.g.Ptxl-LA₂₀₀/Ptxl-LA₁₀₀, or Ptxl-LA₁₀₀/Ptxl-LA₂₀₀. In the above methodsthe relative amounts of polymer conjugate can be varied as desired toobtain particle so desired size and properties.

FIG. 10B is a graph showing the change in particle size of NCs onaddition of different polymer conjugates to the NC to form core-shellNCs.

Particle sizes for FIGS. 10 A and B were analyzed by dynamic lightscattering. More specifically, particle sizes were detected by aZetaPALS dynamic light-scattering (DLS) (15 mW laser, incident beam=676nm; Brookhaven Instruments, Holtsville, N.Y.).

Example 16 Cytotoxicity Test for NCs

PC-3 cells were plated in a 96-well plate for 24 h (10,000 cells perwell). On the day of experiments, cells were washed with prewarmed PBSand different concentration of fresh prepared NCs (prepared in 1×PBS)was added. The control cells were incubated with medium. The cells wereincubated for a total of 24 hours in the incubator at the 5% CO₂atmosphere. After that, the medium was removed and reconstitute with MTTsolution and OptiMEM medium for a further incubation of 3 hours. Theresulting crystals were dissolved and final cell viability was assessedcalorimetrically by microplate reader at 655 nm. The results of certaincytoxicity studies are shown in FIGS. 7A-D for NCs formed with Ptxl,Dtxl, CPT, and Doxo with varying M/I ratios compared to free drug asshown in the figure.

Analogous MTT cytotoxicity tests were performed using NC carryingmultiple drugs in human prostate cancer cells, PC-3 cells, with 72 hoursof incubation. Relative toxicities of a series of NC as measured by IC₅₀is reported in Table 3.

TABLE 3 MTT Cytotoxicity for Multi-Drug Loaded NCs NC IC₅₀ Ptxl-LA₂₅837.22 ± 29.81 Ptxl-LA₂₅/CA-LA₂₅ 622.14 ± 14.61 Ptxl-LA₂₅/CA-LA₁₀ 168.01± 10.79 Doxo-LA₂₅ 286.68 ± 5.65  Doxo-LA₂₅/CA-LA₂₅ 240.16 ± 18.36Doxo-LA₂₅/CA-LA₁₀ 84.89 ± 7.14

As shown in the results of Table 3, the dual drug NC's combining thetaxane (paclitaxel) and cyclopamine or the anthracycline antibiotic(doxorubicin) and cyclopamine exhibit synergistic effect against theprostate cancer cells. With being bound to any particular theory ofactivity, it is believed that inhibition of Shh by cyclopaminesubstantially improved the efficacy of the taxane and anthracyclineantibiotic against cancer cells.

Example 17 Inhibition of the Hedgehog Pathway Using NCs ContainingCyclopamine Assessed Using a Luciferase Assay

Shh-LIGHT2 cells, which stably incorporated Gli-dependent fireflyluciferase and constitutive Renilla luciferase reporters, were culturedto confluency in 96-well plates and then treated with (1) variousconcentrations of cyclopamine carried in PLA-cyclopamine NCs (e.g.,CA-LA₁₀ NCs or CA-LA₂₅ NCs) in DMEM containing 0.5% bovine calf serumwith or without (2) PLA-purmorphamine NEs. FIG. 11A is a graph ofrelative Renilla Luciferase activity as a function of cycloproamineconcentration (microM). The treated cells were then cultured for 36 hunder standard conditions, and firefly and Renilla luciferase activitieswere determined using a dual luciferase kit (Promega) according to themanufacturer's protocols.

Example 18 Effect of Catalyst on Structure of Conjugates

As described above in Example 1, in cases in which the drug or otherchemical species carried more than one functional group that canfunction for initiation of polymerization with a catalyst, the structureor nature of the catalyst can affect which of the functional groups isinvolved in the polymerization and to the site or sites in the drug (orother chemical species) the growing polymer attaches. FIG. 12 shows theeffect of use of different catalysts on the molecular weight andpolydispersity (PDI) of drug-polymer conjugates made by the methodsherein. In a specific case, the figure shows the results of usingdifferent catalysts on conjugate formation with pacitaxel usingLA/Ptxl/catalyst molar ratio of 200/1/1 to prepare Pxtl-LA₂₀₀ ofexpected MW of 29,653. Ptxl formally contains three OH groups (2′-OH,1-OH and 7-OH) that could initiate polymerization of LA. As noted above,however, it is considered less likely for steric reasons that the 1-OHwould be involved in initiation which is believed to involve metal oxideformation with the OH group. It is believed that a source ofpolydispersity in the formation of Pxtl-LA conjugates is the formationof some portion of the conjugates with polymer attached at two sites inthe molecule. As shown in the figure, the actual MW and the PDI asmeasured by standard Gel Permeation Chromatography of Pxtl-LA₂₀₀conjugates varies as a function of the catalyst used. Use of Zn catalystwith bulky ligands (catalysts 4 and 5, from FIG. 12) results inconjugates with lower polydispersity. It is believed that the lowerpolydispersity is associated with increased selectivity for polymerattachment to one site, likely the 2′-OH site in Ptxl.

Example 19 Determination of MW and PDI of Drug-Polymer Conjugates UsingGel Permeation Chromatography

Drug-polymer conjugates were prepared as described in previous examplesemploying the drug as the initiator (in the presence of catalyst) forpolymerization of LA. Actual MW and PDI were measured using well-knownmethods of Gel Permeation Chromatography.

TABLE 4 GPC data of Nanoconjugates containing Ptxl, CPT, Dtxl, Doxo, CAor CP NC Expected MW Actual MW PDI Ptxl-LA₂₀₀ 29653 28160 1.021Ptxl-LA₁₅₀ 22453 22490 1.032 Ptxl-LA₁₀₀ 15253 14030 1.043 Ptxl-LA₅₀ 80539658 1.040 Cpt-LA₂₀₀ 29148 30440 1.176 Cpt-LA₁₀₀ 14748 20080 1.207Cpt-LA₇₅ 11148 12670 1.173 Cpt-LA₅₀ 7548 8978 1.257 Cy5-LA₃₀₀ 4370644550 1.098 Cy5-LA₂₀₀ 29306 30790 1.213 CA-LA₁₅₀ 22011 22620 1.166CA-LA₁₀₀ 14811 19800 1.102 Dtxl-LA₃₀₀ 44006 51350 1.045 Dtxl-LA₂₀₀ 2960630600 1.095 Dtxl-LA₁₀₀ 15206 20700 1.086 Dtxl-LA₅₀ 8006 12090 1.169CP-LA₂₀₀ 30002 40150 1.161 CP-LA₁₀₀ 15602 31810 1.324 CP-LA₅₀ 8402 214401.337 Doxo-LA₂₀₀ 29343 40630 1.156 Doxo-LA₁₀₀ 14943 19400 1.253

The forgoing examples are illustrative and in no way are intended tolimit the scope of the invention.

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1. A method for preparing particles for in vivo delivery of a drug whichcomprises the steps of: (a) providing a drug the structure of whichcomprises one or more hydroxyl or thiol groups; (b) conductingring-opening polymerization of one or more cyclic monomers selected fromcyclic esters, cyclic carbonates, cyclic phosphate, cyclic silicone,cyclic peptides or amino acid derivative, or cyclic phosphazane, or acombination thereof in anhydrous, water-miscible solvent in the presenceof the drug as a polymerization initiator and a polymerization catalystto form a covalent drug-oligomer or drug-polymer conjugate; and (c)forming particles comprising the drug-oligomer or drug-polymer conjugateranging in size from 2 nanometers to 100 microns.
 2. The method of claim1 wherein the particles are nanoparticles having particle size rangingfrom 2 nm to 300 nm.
 3. The method of claim 1 wherein the particles aremicroparticles ranging in particle size from 0.200 micrometers to 400micrometers.
 4. The method of claim 1 further comprising the step ofmodifying the surface of the particle.
 5. The method of claim 4 whereinthe particle surface is modified by providing a coating layer of apolymer which may be the same or different from that of the conjugate.6. The method of claim 5 wherein the particle surface is modified withsurface pegylation with PEG chain length ranging from PEG 400 to PEG40,000.
 7. The method of claim 6 wherein the surface pegylation isprovided by forming covalent particle-PEG linkages.
 8. The method ofclaim 6 wherein the surface pegylation is provided through non-covalentinteractions using hydrophobic polymer-b-PEG.
 9. The method of claim 4wherein the surface of the particle formed is modified with ahydrophilic or hydrophobic surface modifier.
 10. The method of claim 4wherein the surface of the particle formed is modified with anamphiphilic polymer.
 11. The method of claim 10 wherein the amphiphilicpolymer comprises PEG.
 12. The method of claim 10 wherein theamphiphilic polymer is a copolymer comprising PEG.
 13. The method ofclaim 9 wherein the surface of the particle formed is modified bycovalent or non-covalent attachment to one or more of a peptide,protein, saccharide, carbohydrate, nucleic acid or a combinationthereof.
 14. The method of claim 1 wherein the terminal groups of thepolymer or oligomer are selected from the group consisting of hydroxyl,thiol, amine, azide, alkyne, alkene, ketone, phenol, halides, imidazole,guanidinium, carboxylate, or phosphate groups.
 15. The method of claim14 wherein the surface of the particle formed is modified by coating orconjugation of one or more targeting ligands.
 16. The method of claim 1wherein the particles have a drug concentration gradient.
 17. The methodof claim 1 wherein the particles have a multilayer structure withdifferent drug concentrations or different types of drugs in differentlayers.
 18. The method of claim 17 wherein at least one layer of theparticles is formed from the drug-oligomer or drug-polymer conjugate.19. The method of claim 1 wherein the drug is a drug selected from thedrugs in FIG.
 13. 20. The method of claim 1 wherein the drug is a drugselected from Darunavir (TMC-114), Tipranavir (TPV), Saquinavir (SQV),Ritonavir (RTV), Indinavir, Nelfinavir (NFV), Amprenavir (APV),Lopinavir (ABT-378), Atazanavir (ATV), Vinorelbine bitartrate,fulvestrant, Sarcodictyins, camptothecins, Vinblastine, bryostatin 1,(+)-Cylindricine, (+)-Lactacystin, Aeruginosin 298-A, (+)-Fostriecin,Garsubellin A/Hyperforin, (S)-Oxybutynin, Epothilone A, Zidovudine(AZT), Lamivudine (3TC), Didanosine (ddI), Abacavir (ABC), Emtricitabine(FTC), bamethane, ethamivan, hexachlorophene, salicylanilide,pyrocatechin, thymol, pentazocine, phloroglucinol, eugenol, niclosamide,terbutaline, dopamine, methyldopa, norepinephrine, eugenol, α-naphthol,polybasic phenols, adrenaline, dopamine, phenylephrine, metaraminol,fenoterol, bithionol, alpha-tocopherol, isoprenaline, adrenaline,norepiniphrine, salbutamol, fenoterol, bithionol, chlorogenicacid/esters, captopril, amoxicillin, betaxolol, masoprocol, genistein,daidzein, daidzin, acetylglycitin, equol, glycitein,iodoresiniferatoxin, SB202190, or tyrphostin SU1498.
 21. The method ofclaim 1 wherein the nanoparticles formed range in size on average from 2nm to 400 μm.
 22. The method of claim 1 wherein the catalyst is anorganometallic catalyst or an organo catalyst of a ring-openingpolymerization reaction.
 23. The method of claim 1 wherein thepolymerization reaction is performed in a solvent selected from THF,acetone, methylene chloride, chloroform, dimethylformamide, DMSO,acetonitrile or mixtures thereof.
 24. The method of claim 1 whereinnanoparticles are formed by combining a solution of the covalentdrug-polymer conjugate or the drug-oligomer conjugate in awater-miscible solvent with an excess of water.
 25. The method of claim1 wherein the molar ratio of cyclic monomer to drug initiator rangesfrom 5000/1 to 2/1.
 26. The method of claim 1 wherein the drug is ahydroxyl-containing small organic molecule.
 27. The method of claim 1wherein the drug is a macromolecule.
 28. The method of claim 1 whereinthe drug is a peptide, saccharide or nucleic acid.
 29. The method ofclaim 1 wherein the cyclic monomer is a cyclic ester, cyclic carbonateor a combination thereof.
 30. The method of claim 1 wherein the catalystis an orgaometallic catalyst.
 31. A covalent drug-oligomer ordrug-polymer conjugate prepared by polymerizing one or more cyclicmonomers selected from the group consisting of cyclic esters, cycliccarbonates, cyclic phosphates, cyclic siloxanes, cyclic peptides oramino acid derivative, or cyclic phosphazanes, in the presence of a drugthe structure of which comprises one or more hydroxyl groups and aring-opening polymerization catalyst wherein the drug comprising the oneor more hydroxyl groups is the initiator of the polymerization reaction.32. The covalent drug-polymer conjugate of claim 31 wherein the oligomercomprises 5000 or fewer repeating units of the ring-opened monomer. 33.The covalent drug-polymer conjugate of claim 31 wherein the oligomercomprises 500 or fewer repeating units of the ring-opened monomer. 34.The covalent drug-oligomer conjugate of claim 31 wherein the oligomercomprises 50 or fewer repeating units of the ring-opened monomer. 35.The covalent drug-oligomer conjugate of claim 31 wherein the oligomercomprises 20 or fewer repeating units of the ring-opened monomer. 36.The covalent drug-oligomer conjugate of claim 31 wherein the oligomercomprises 10 or fewer repeating units of the ring-opened monomer.
 37. Apharmaceutical composition comprising particles made by the method ofclaim
 1. 38. A pharmaceutical composition comprising particles made froma covalent drug-oligomer or drug-polymer conjugate of claim
 31. 39. Ananoparticle comprising a core/shell structure or a multiple layerstructure wherein at least one of the core or shell or one of the layersis formed from a drug-polymer or a drug-oligomer conjugate which isprepared by conducting ring-opening polymerization of one or more cyclicmonomers selected from cyclic esters, cyclic carbonates, cyclicphosphate, cyclic silicone, cyclic peptides or amino acid derivative, orcyclic phosphazane, or a combination thereof in an anhydrous,water-miscible solvent in the presence of the drug as a polymerizationinitiator and a polymerization catalyst to form the drug-oligomer ordrug-polymer conjugate.
 40. A method for delivery of a drug to anindividual in need thereof which comprises administering to theindividual nanoparticles of claim 39 and which comprise the drugcovalently conjugated to an oligomer or polymer comprising ring-openedmonomer repeating units.
 41. The method of claim 40 wherein the monomersare selected from lactides, glycolides or a combination thereof.
 42. Amethod for delivery of a drug to an individual in need thereof whichcomprises administering to the individual nanoparticles prepared by themethod of claim 1 and which comprise the drug covalently conjugated toan oligomer or polymer comprising ring-opened monomer repeating units.43. A method for making a medicament comprising a drug whose structurecomprises one or more hydroxyl groups or thiol groups which comprisescovalently attaching the drug to an oligomer or polymer formed byrein-opening polymerization by introducing the drug into thering-opening polymerization reaction to function therein as theinitiator of polymerization.
 44. A method for preparing particles for invivo delivery of a chemical species which has at least one hydroxylgroup or thiol group which comprises the steps of: (a) conductingring-opening polymerization of one or more cyclic monomers selected fromcyclic esters, cyclic carbonates, cyclic phosphate, cyclic silicone,cyclic peptides or amino acid derivative, or cyclic phosphazane, or acombination thereof in anhydrous, water-miscible solvent in the presenceof the chemical species having at least one hydroxyl group as apolymerization initiator and a polymerization catalyst to form acovalent drug-oligomer or drug-polymer conjugate; and (c) formingparticles comprising the drug-oligomer or drug-polymer conjugate rangingin size from 2 nanometers to 100 microns.
 45. A covalent oligomer orpolymer conjugate prepared by polymerizing one or more cyclic monomersselected from the group consisting of cyclic esters, cyclic carbonates,cyclic phosphates, cyclic siloxanes, cyclic peptides or amino acidderivative, or cyclic phosphazanes, in the presence of a chemicalspecies the structure of which comprises one or more hydroxyl groups anda ring-opening polymerization catalyst wherein the drug comprising theone or more hydroxyl groups is the initiator of the polymerizationreaction.
 46. The conjugate of claim 45 wherein the chemical species isselected from the groups consisting of a diagnostic reagent, a peptide,a saccharide, a carbonhydrate, an inorganic species, a contrast agent, avitamin, a nutrient, a nucleic acid, an RNA molecule, an siRNA, or a DNAmolecule.